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Departments of Neuroscience (R.P., A.M.R., E.T.) and Internal Medicine (P.B.), University of Tor Vergata, 00133 Rome; the Department of Cytomorphology, University of Cagliari (G.-L.F.), 09124 Cagliari; and the Institute of Neurobiology, National Research Council (R.P., A.M.R., E.T., A.L.), 00137 Rome, Italy
Address all correspondence and requests for reprints to: Dr. Roberta Possenti and Andrea Levi, Institute of Neurobiology, National Research Center, Via Carlo Marx 15, 00137 Rome, Italy. E-mail: r.possenti{at}in.rm.cnr.it alevi@in.rm.cnr.it.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vgf, a gene initially identified as a nerve growth factor (NGF)-responsive gene in the PC12 cell line (16, 17), is specifically expressed in subpopulations of neurons and in a subset of neuroendocrine cells (18, 19). Although the physiological functions of the vgf gene products await clarification, a hint regarding their possible function may come from their intracellular localization. The VGF peptides are stored in dense core granules and are released in response to secretory stimuli, suggesting a neuromodulatory function. Recently, transgenic mice bearing vgf gene disruption were described, showing neonatal growth retardation, feeding abnormalities, and hypermetabolic state (20) as well as altered mating behavior and reduced fertility (21). Such findings may be consistent with an essential role of VGF in the hypothalamus-pituitary-gonadal axis, which would fit well with the abundance of VGF products in female rat gonadotrope and lactotrope cells and their considerable plasticity along the estrous cycle and after gonadectomy (22).
The primary translation product of vgf is a protein of apparent 90 kDa that in neurons is cleaved by the prohormone convertases PC1/3 and PC2 in a late compartment of the secretory pathway (Trani, E., N. Canu, A. M. Rinaldi, G.-L. Ferri, R. Possenti, and A. Levi, manuscript in preparation). The most abundant maturation products of VGF are polypeptides of 20, 18, and 10 kDa, respectively named VGF-20, VGF-18, and VGF-10 that are stored in dense core granules of neuronal and endocrine cells (22) and secreted upon cell depolarization (23). Such peptides are likely candidates for physiologically active species, endowed with neuromodulatory functions.
We have surveyed different insulinoma cell lines, ßTC-6, INS-1, RIN 1046–38, and HIT-T15, as well as primary cultures of pancreatic islets from 6-day-old rats. All of them express vgf messenger RNA (mRNA) (15, 24). We have chosen to further characterize the insulinoma cell line INS-1, a well established model system for studying ß-cell functions (25).
The aim of this study was to investigate modulation of the expression of vgf gene in the insulinoma cell line INS-1. In addition, we analyzed the processing and secretion of VGF mature peptides in response to stimuli that modulate secretion from pancreatic ß-cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The PC12 cell line was originally obtained from Dr. L. A.
Green, RIN 1046–38 was obtained from Dr. C. Montrose-Rafizadeh,
ßTC-6 cells were provided by Dr. S. Efrat, and HIT-T15 cells were
provided by Dr. C. B. Wallheim.
The INS-1 cell line (25) was obtained from the laboratory of Dr. C. B. Wollheim at passage 72 and was used until passage 95 (at this passage the cells still retain the functional insulin secretion response to a physiological concentration of glucose). Cells were grown, as described, in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES (pH 7.4), and penicillin-streptomycin (100 U/ml and 0.1 mg/ml, respectively). The cells were incubated at 37 C in a humidified atmosphere with 5% CO2. Phorbol 12-myristate 13-acetate (PMA) was added to a final concentration of 100 nM. (Bu)2cAMP was used at 1 mM. Addition of forskolin at 10 µM plus isobutylmethylxanthine at 100 µM gave the same results as those obtained with (Bu)2cAMP, and these two treatments were used interchangeably. The fungal metabolite brefeldin A, an inhibitor of intracellular trafficking, was used at 2 µg/ml.
Pancreas islet immunohistochemistry
Samples of pancreas (adjacent to duodenum-jejunum or from body
and tail regions) were taken from normal rats (males and females;
Sprague Dawley, Wistar, and Long Evans; n = 8), killed by ether
overdose or decapitation. Procedures were approved by the local ethical
committee and conformed to European Union regulations. Samples were
immersion fixed in 4% paraformaldehyde (3–8 h), washed in 7–10%
sucrose-PBS (3–16 h), and frozen for cryosectioning (6–10 µm).
After the primary incubation (1:2,000–10,000 dilution, overnight) with anti-VGF609–617, sections of pancreas were treated with an affinity-purified, donkey, biotinylated, antirabbit IgG preparation (for 1 h), followed by Cy3-conjugated avidin (both from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Blocking with unconjugated avidin and biotin was used to reduce background staining (Vector Laboratories, Inc., Burlingame, CA). Where appropriate, sections were also treated with guinea pig antiinsulin serum (Biodesign, Kennebunk, ME), followed by donkey fluorescein isothiocyanate (FITC)-conjugated antiguinea pig IgG antibodies (Jackson ImmunoResearch Laboratories, Inc.). Routine staining controls included substitution of each layer, in turn, with PBS as well as the use of nonimmune rabbit serum. Sections of pancreas slices were observed using an Olympus Corp. BX-60 microscope (Olympus Corp. Italia, Milan, Italy) equipped with custom-designed filter systems (produced to our specification by Chroma, Brattleboro, VT), which completely prevented cross-talk of FITC and Cy3 in visual observation (26). To provide maximum efficiency, the Cy3 filter system was centered on the yellow emission component of this fluorochrome; hence, Cy3 was shown as a bright golden-yellow signal. E100-SW film (Eastman Kodak Co., Rochester, NY) and a PM-30 system (Olympus Corp.) were used for photography.
Immunofluorescence
INS-1 cells were grown on poly-L-lysine coated
coverslides, washed three times with PBS, fixed with 4%
paraformaldehyde in PBS for 10 min, and permeabilized with 0.2% Triton
X-100 in 50 mM Tris-HCl, pH 7.5, for 10 min. Cells were
incubated with anti-VGF573–617 serum (1:2000 dilution for
1 h) and revealed with rhodamine isothiocyanate (TRITC)-conjugated
goat antirabbit antibodies. Permeabilized cells were incubated with
guinea pig antiinsulin serum (1:1000 dilution for 1 h) and stained
with FITC-conjugated goat antiguinea pig antibodies. Confocal
microscopy was carried out with a Leica Corp.
(Heidelberg, Germany). TCS 4D system equipped with a 100 x
1.3–0.6 oil immersion objective. Images of double labeled samples were
recorded with simultaneous excitation and detection of both dyes to
ensure proper image alignment. To correct for possible cross-talk
resulting from overlapping excitation and emission spectra of the dyes
used, recorded images were corrected when necessary using the
MultiColor analysis package software from Leica Corp. and
compared with images recorded for single dye excitation and detection
(27).
Northern blot analysis
Total RNA was prepared by the guanidinium thiocyanate method
(28). Northern blot analysis was performed using as a probe a 1-kbp
BamHI fragment from rat vgf complementary DNA.
The amount of RNA was normalized by reprobing the same filter with a
0.6-kbp HpaII fragment from a mouse histone H4 complementary
DNA.
Hybridization and washing were performed according to standard procedures. The Northern blots were exposed and analyzed using a Molecular Dynamics, Inc. PhosphorImager 400A (Sunnyvale, CA) with ImageQuant version 3.2 software. Data from two different experiments were averaged.
Cell transfection for chloramphenical acetyltransferase (CAT)
assays
INS-1 cells were transfected with a vgf promoter
reporter plasmid, previously described in detail (29). In this plasmid
a 0.8-kbp long fragment, derived from the 5'-vgf promoter
region, drives the transcription of the CAT reporter (VGF-CAT) in a
tissue-specific, NGF- and cAMP-inducible manner. CAT gene driven by
Rous sarcoma virus long terminal repeat (RSV-CAT) was used as a
control. Briefly, the cells were plated the day before transfection at
50% confluence in 10-cm tissue culture dishes. Transfection was
performed with lipofectamine reagent (Life Technologies)
according to the manufacturer’s protocol. Twenty-four hours after
transfection, the cells were divided into 10 35-mm dishes and exposed
to the different agents; after an additional 48 h the cells were
collected, and equal amounts of proteins were used for CAT assay as
previously described (30). Data from different experiments (five for
VGF-CAT and three for RSV-CAT, performed in duplicate) were
averaged.
Immunoblotting
Cells were harvested in PBS and directly lysed in SDS sample
buffer. Extracts were subjected to SDS-PAGE on a 10–18%
polyacrylamide gradient. Proteins were transferred to nitrocellulose
membrane (Amersham). The amount of protein loaded was
verified by Ponceau protein detection (Sigma Chemical Co.). The membranes were blocked with 3% low fat milk in 10
mM Tris (pH 8), 150 mM NaCl, and 0.05%
Tween-20 and incubated with VGF609–617 (1:5000 dilution).
After extensive washing, the filters were incubated with horseradish
peroxidase-conjugated protein A and developed with the ECL system
following the manufacturer’s instructions. Scans of ECL-developed
films were analyzed by Bio-Rad densitometer model 620 and quantified by
Bio-Rad Program Manager software (Bio-Rad Laboratories, Inc., Hercules, CA).
Expression of the NGF receptors, the low affinity p75 and the high affinity trk-A, was detected in PC12 and INS-1 cells by wheat-germ agglutinin agarose precipitation and Western blot analysis. Approximately 107 cells were lysed in lysis buffer [20 mM Tris-Cl (pH 8), 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 0.5 mM phenylmethylsulfonylfluoride, 1 µg/ml aprotinin, and 10 µg/ml leupeptin]. Glycoproteins were absorbed to a 1:1 suspension of wheat-germ agglutinin agarose in lysis buffer and incubated for 4 h at 4 C. After separation on 7.5% SDS-PAGE gels, the proteins were transferred to nitrocellulose membrane and revealed with rabbit anti-p75 antibodies, obtained from Dr. M. Chao, and rabbit anti-trk-A antibodies, provided by Dr. L. F. Reichardt.
Stimulation of cell secretion
The regulated secretion of VGF peptides was measured in two
different milieu according to the secretory stimuli employed: the
response to different concentrations of glucose was measured in
modified Krebs-Ringer-bicarbonate-HEPES buffer (KRBH buffer: 134
mM NaCl, 4.7 mM KH2PO4, 1.2
mM MgSO4, 1 mM CaCl2,
and 10 mM HEPES, pH 7.4, without BSA). The responses to PMA
and to an intracellular increase in cAMP were measured in serum-free
RPMI. The cells were washed three times in serum-free medium for 10 min
each time. Fresh medium containing the different secretagogues was
applied and collected after 3 h of stimulation. A preliminary
experiment demonstrated no adverse effect on cell metabolism due to
nutrient deprivation for such a time interval. The medium was
centrifuged for 5 min at 3000 x g to remove any
detached cells, and the secreted proteins were then precipitated by the
addition of trichloroacetic acid at a 15% final concentration and
examined by Western blot analysis.
Insulin release was measured as described by Herbert et al.
(31). Briefly, the cells were seeded in culture medium in 24-multiwell
plates at a density of 105 cells/ml. After 48 h of
growth, cells were washed twice at 37 C for 30 min each time with a
glucose-free buffer containing 0.1% RIA grade BSA. Thereafter, cells
were incubated for 1 h in the same buffer in the absence or
presence of glucose, (Bu)2cAMP, or PMA as
indicated in Table 1
. Aliquots of the
supernatant were collected and stored at -20 C for insulin RIA. Cells
were extracted overnight at 4 C with a solution of acidified ethanol
for assay of the intracellular insulin content. Insulin determination
was performed by the dextran-charcoal method as previously described
(31), using antiinsulin antibodies raised in guinea pig, porcine
insulin standard, and [125I]insulin (NEN, Boston, MA).
Two different experiments were run in quadruplicate and counted in
triplicate.
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Results |
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, thick
arrow), and the 20-, 18-, and 10-kDa peptides (Fig. 1, thin
arrows). These low molecular mass species represent the mature
products that are detected in extracts of primary cultures of
cerebellar granule cells and are absent in PC12 cells (23). By RT-PCR
we also detected vgf mRNA in primary cultures of pancreas
islets and in the hamster-derived insulinoma cell line HIT-T15 (24). In
this cell line we could not show immunoreactivity, probably due to the
low affinity of the antisera raised against the rat protein with the
hamster species (not shown).
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). Double
immunostaining confirmed that VGF immunoreactivity colocalized with
insulin in most cases (Fig. 2, B vs. C). VGF-immunoreactive
nerve fibers were also visible and were apparently more abundant than
in the surrounding parenchyma (Fig. 2, A and B). Confocal studies of
INS-1 cells stained with anti-VGF573–617 serum resulted in
a speckled localization of the immunofluorescent signal that is
characteristic of secretory vesicles (Fig. 2D). These were the most
abundant in a perinuclear region typical for the Golgi apparatus (Fig. 2G). Double immunostaining for insulin revealed a colocalized signal
(Fig. 2, E and H) in most granules. Thus, when VGF and insulin
immunostaining were shown as green and red (respectively) in the
digital double images, the large majority of granules showed a
composite yellow label, indicative of colocalization (Fig. 2, H and
I).
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and
demonstrated that elevation of intracellular cAMP leads to a 6- to
8-fold increase in vgf mRNA. Activation of protein kinase C
(PKC) with PMA or a high level of extracellular glucose (20
mM) showed no effect on vgf mRNA level in INS-1
cells (Fig. 3A). As previously described (15), we did not observe any
significant effect of NGF on vgf mRNA levels in INS-1 cells.
This may due to the low level of expression of the high (trk-A) and low
(p75) affinity of NGF receptors. As measured by Western blots (Fig. 3B), these receptors are expressed in INS-1 cells less than 10- to
100-fold compared with those in PC12 cells.
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, the
vgf promoter was stimulated about 3-fold by cAMP. No
induction of the RSV-promoter was observed in parallel experiments,
ruling out a possible nonpromoter-specific general activation of
transcription by elevated cAMP. Stimulation of the transfected
vgf promoter required an intact cAMP response element (CRE)
element located 80 bp 5' to the transcription start, as deletion of
this element negated the induction by cAMP (not shown). Neither NGF nor
PMA had any significant effect on basal or cAMP-stimulated
transcriptional activity of vgf promoter.
VGF protein is synthesized and proteolytically processed in INS-1
cells
For the level of vgf mRNA, treatment for 16 h with
(Bu)2cAMP (Fig. 4A, lanes 3,
5, 7, and 8), but not with NGF and/or PMA (Fig. 4A lanes 2, 4, and 5),
increased the amount of VGF peptides. When cells were exposed to
brefeldin A, a fungal metabolite that inhibits the anterograde
vesicular traffic from the endoplasmic reticulum to the Golgi,
processing of the 90-kDa VGF (thick arrow) into the lower
molecular mass species (thin arrows) was prevented, as shown
in Fig. 4B. Inhibition of processing of VGF was visible after 6 h
(compare lane 2 with lane 4) and was prominent after 16 h (lanes 3
and 5). These results demonstrate that the proteolytic cleavage of the
VGF precursor form occurs in a late compartment of the secretory
pathway.
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. The amount of the
90-kDa form steadily increased and reached a maximum between 16–24 h
of (Bu)2cAMP treatment with a 10-fold increase with respect
to the level in untreated INS-1 cells. Compared with that at time zero,
we observed an initial decrease in the quantity of the lower molecular
mass species after 1 and 3 h (Fig. 4C, lanes 2 and 3). This is the
consequence of preferential depletion of the VGF-20, VGF-18, and VGF-10
species with respect to the VGF precursor form due to a secretory
stimulus provided by cAMP (see next section).
VGF peptides are released from INS-1 cells in response secretory
stimuli
To study the regulated secretion of VGF species, INS-1 cells were
treated overnight with 1 mM (Bu)2AMP, washed
extensively, and then exposed to secretory stimuli in either serum-free
RPMI or KRBH buffer. Qualitatively similar results were obtained
without overnight treatment with (Bu)2cAMP, which was used
for convenience because it resulted in a strong increase (>10-fold) in
the intracellular level of VGF (compare lanes 1 and 5 in Fig. 4C).
Figure 5 shows an example of Western blot
analysis of anti-VGF-reactive polypeptides in cell extracts and media
after 3-h treatment of INS-1 cells with 1 mM
(Bu)2cAMP and 100 nM PMA in serum-free RPMI.
Both treatments resulted in the preferential release into the medium of
VGF-20, VGF-18, and VGF-10 relative to the high molecular mass form
(Fig. 5A, lanes 4–6) and resulted in a parallel decrease in their
intracellular levels (Fig. 5A, lanes 1–3). No cell lysis was
observed during the depolarization stimuli, as confirmed by the absence
of any detectable actin in the extracellular medium (not shown).
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shows the
ratio between the quantity of the low molecular mass peptides (VGF-20,
VGF-18, and VGF-10) secreted upon stimulation and the quantity of the
constitutively released ones. Activation of protein kinase A (PKA) and
protein kinase C (PKC) stimulated the secretion of VGF peptides about
3- and 6-fold, respectively. Up to 40% of the total content of VGF-20,
VGF-18, and VGF-10 was released within 3 h in response to PMA. By
comparison, less than 5% of the initial amount of low molecular mass
VGF species were released into the medium in the absence of
stimulation. The high molecular mass form of VGF (thick
arrow) was also secreted, albeit to a much lower extent. The
different ratios between the low and the high molecular species in the
medium and in the cell extract (7.5 and 1.5, respectively) further
supported the absence of cell lysis and suggested that the low
molecular mass species are the ones preferentially secreted. Elevation
of intracellular cAMP and addition of PMA had lower stimulatory effect
in KRBH buffer (not shown), confirming that they were fully effective
only when cooperating with other secretagogue nutrients such as
glucose, glutamine, and other amino acids (extensively discussed in
Refs. 32, 33). Stimulated secretion of VGF peptides was also
observed in response to membrane depolarization with high extracellular
potassium, but no significant secretory effect was observed upon NGF
stimulation (data not shown).
Among the different insulinoma-derived cell lines, INS-1 cells, which
retain the well characterized differentiative state of ß-cells,
display a dose-dependent insulin release after exposure to a
physiological range of glucose, cAMP, or PMA (25, 33). INS-1 cells were
therefore stimulated for 3 h with glucose ranging from 0–30
mM in KRBH buffer, and the amount of secreted VGF low
molecular mass peptides was semiquantified by densitometric analysis of
Western blots. Figure 6A shows the ratio
between the amounts of VGF peptides released in response to different
concentrations of glucose, and the amounts released in the absence of
glucose. The maximum stimulation was achieved between 10–15
mM glucose and caused a 6-fold increase over the basal
constitutive level. In this condition up to 15% of total VGF initially
present inside the cells was released into the medium. As show in Fig. 6B, secretion of VGF peptides in response to glucose was further
stimulated by cAMP similarly to what have been described for insulin
(25, 33).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the insulinoma-derived cell line INS-1, vgf expression is regulated by cAMP, and VGF protein undergoes a very similar maturation process, previously shown to occur in fully differentiated neurons (23). vgf is a tissue-specific gene whose cell type-restricted transcription is controlled by both positive and negative regulatory elements within 2 kbp 5' to the transcription initiation site (29, 34). It has been shown that vgf expression is induced by elevation of intracellular cAMP in INS-1 cells, which is related to the presence of a CRE element located at -80 from the transcription initiation site. We have previously shown that the CRE element is functional in a cell type-restricted way, as elevated cAMP activates vgf in neuronal cells but fails to do so in nonneuronal cells (34). The CRE was previously shown to be necessary for NGF induction of vgf transcription (35, 36), and here we have demonstrated that the CRE in itself is not sufficient, as in INS-1 cells, NGF failed to up-regulate the endogenous and transfected vgf promoters. It is possible that insulinoma cells lack part of the transduction machinery for NGF; alternatively, the expression of the NGF receptors may be too low to elicit a full biological response. The steady state level of vgf mRNA, as measured by Northern blot, is increased after 16 h by approximately 6-fold in response to elevated intracellular cAMP. By comparison, the transcriptional induction of the transfected promoter is about 3-fold. This may indicate either that motifs important for the induction are present outside the promoter region used in this study or that the stability of vgf mRNA is increased by elevation of cAMP.
We have previously shown that proteolytic maturation of VGF occurs in a cell type-restricted way; adrenal medulla and the derived cell line PC12 contain almost exclusively the high molecular mass forms (90 and 80 kDa). Conversely, the low molecular mass forms, VGF-20, VGF-18, and VGF-10, appear to be present in varying proportions in different neuronal populations (23). Much evidence suggests that maturation of VGF is the consequence of proteolytic cleavage by the tissue-specific prohormone convertases PC1/3 and PC2 (Trani, E., et al., manuscript in preparation). These convertases have been detected in insulinomas and pancreatic islets and are responsible for processing proinsulin (37). They are fully active in late compartments of the regulated secretory pathway. This is consistent with the inhibitory action of brefeldin A on VGF maturation. It is presently unknown whether the high molecular mass forms are endowed with a specific function other than being the precursors for the low molecular mass VGF polypeptides. These are considered the ones that might exert neuromodulatory functions upon secretion from neuronal cells. Here we demonstrate that in INS-1 cells, VGF-20, VGF-18, and VGF-10 are also released through the regulated secretory pathway. Pancreatic ß-cells are capable of regulated secretion in response to a number of stimuli. Glucose, amino acids, potassium depolarization, and activation of either PKA or PKC were shown to promote the secretion of insulin and neuropeptide Y (reviewed in Ref. 38). Activation of PKA and PKC is also responsible for VGF-derived peptides secretion, suggesting that the mature forms of VGF and insulin are stored in the same type of vesicles, as also shown by immunofluorescence costaining. Alternatively, different dense core secretory granules could be present in insulinoma cells and fusion with the plasma membrane of insulin-containing and VGF-containing vesicles may be controlled by the same transduction machinery (38). Electron microscopy studies are necessary to unequivocally establish this point.
The presence of VGF in endocrine cells and in nerve fibers of rat pancreatic islets and the regulated secretion of the VGF products in INS-1 cells suggest that these peptides, besides their role in the function of the hypothalamic/pituitary-axis, are involved in the function of the pancreas islets.
We are attempting to investigate the possible effect of VGF peptides in primary cultures of pancreas islets as well as in different pancreas-derived cell lines.
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Acknowledgments |
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Footnotes |
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Received August 10, 1998.
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