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Department of Histology and Medical Embryology (F.N., M.P., S.M.), University "La Sapienza," and Istituto Dermopatico dell’Immacolata (M.P., S.M.), Rome, Italy; Beth Israel Deaconess Medical Center (D.L.G., S.R.G.), New England Baptist Bone and Joint Institute, Harvard Medical School, Boston, Massachusetts; Department of Rheumatology (P.O.), INSERM, Unit 349, Lariboisiere Hospital, Paris, France; Department of Experimental Medicine (A.T.), University of L’Aquila, Italy
Address all correspondence and requests for reprints to: Anna Teti, Ph.D., Department of Experimental Medicine, via Vetoio, Coppito 2, 67100 L’Aquila, Italy. E-mail: teti{at}univaq.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but not the 
isoenzyme, was cytosol-to-membrane
translocated by approximately 50% after 20 min exposure to calcitonin,
whereas protein kinase-C
, which was approximately 40%
membrane-linked in unstimulated cells, translocated by approximately
19%. The human calcitonin receptor expressed by BIN-67 ovary tumor
cells, although displaying higher affinity for calcitonin, failed to
activate phospholipase D and protein kinase-C in response to the
hormone. This receptor lacks the G protein binding consensus site due
to the presence of a 48-bp cassette encoding for a 16-amino acid insert
in the predicted first intracellular loop. This modification is likely
to prevent the calcitonin receptor from associating to
phospholipase-coupled signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of the functional properties of the porcine CTR, cloned from the LLC-PK1 renal tubule cells, revealed that it was coupled to at least two signal transduction pathways. Coupling to the activation of adenylyl cyclase/cAMP/protein kinase A pathway has been described in several cell types (11), and further studies revealed that the same receptor is also coupled to activation of phosphoinositide (PtdIns)-dependent phospholipase C (PLC), which results in Ca2+ mobilization (12), and protein kinase C (PKC) activation (13). The two signal transduction pathways require the cholera toxin sensitive Gs, and the pertussis toxin-sensitive Gi and possibly the Gq PKC-coupled GTP-binding proteins, respectively. Coupling of CTR to these distinct G proteins leads to opposite biological effects associated with e.g. regulation of the sodium pump (i.e. stimulation by protein kinase A and inhibition by PKC) (13). Furthermore, selective activation of the pathways can occur in a cell cycle-dependent manner because Gs is activated in G2 phase and no longer stimulated in S phase (13, 14). Conversely, in LLC-PK1 CTR couples to Gi in S phase, but not in G2 phase (13, 14).
Full activation of multiple signal transduction pathways is not a common feature of all CTR isoforms. In fact, the BIN-67 human ovary tumor cell line expresses a high copy number of a CTR isoform, which is distinguished from the porcine CTR by the presence of a 48-bp cassette which encodes a 16 aa insert in the predicted first intracellular loop (48+) (7). Previous studies by our laboratories and others have shown that this isoform binds CT with high affinity, but fails to stimulate Ca2+ mobilization (7, 9). The functional capacity to activate alternate signaling pathways may provide a mechanism by which CT binding results in different biological responses. Therefore, the dissection of multiple intracellular signals elicited by the same receptor is relevant to fully elucidating the mechanism of action of the hormone.
This study was aimed at investigating whether CT stimulates novel
CTR-dependent signal transfer in LLC-PK1 and BIN-67 cells.
To this end, we focused on membrane phosphatydilcholine (PtdCho)
metabolism and membrane translocation of PKC isoforms induced by
binding of salmon CT (sCT). Our results provide evidence that
phospholipase D (PLD) and PKC, and possibly , isoenzymes are
potential components of CTR(48-)- but not of the CTR(48+)-coupled cell
signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell cultures
LLC-PK1 cells are a porcine kidney epithelial cell
line previously characterized in our laboratory (15). BIN-67 cells are
a human ovary tumor cell line (7). Cells were cultured in DMEM
supplemented with 10% FBS and antibiotics and fed once a week. At
confluence, cells were split 1:20 by standard trypsin procedures.
Radiolabeling and stimulation
[3H]inositolphosphate (InsP) and
[3H]choline labeling. Cells were grown in 12-well
tissue culture plates. On the second day of culture the medium was
substituted with fresh medium containing either 185 kBq/ml
D-myo-[3H]inositol or 74 kBq
[3H]choline chloride and cultured for an additional
48 h.
Fatty acid labeling. Cells were plated into 6-cm tissue culture dishes and grown to 80% confluence. Monolayers were then incubated for 3 h with 9.25 kBq/ml [9,10(n)]-[3H]myristic acid.
After labeling, the cells at 80% confluence (
2 x
105 cells/dish for InsP and [3H]choline
labeling and approximately x 106 cells/dish for fatty
acid labeling) were extensively washed with DMEM supplemented with 20
mM HEPES, pH 7.4, and 0.1% BSA, and stimulated with the
agonist. Incubations were stopped by aspirating the medium and adding
ice-cold methanol for PtdCho, or 10% trichloroacetic for InsP
determination (16). For soluble PtdCho metabolites, stimulation was
terminated by direct addition of 1 vol of ice-cold methanol to the
incubation medium (17).
Measurement of InsP accumulation
[3H]InsP-labeled mono and bisphosphate were
extracted, separated by ion exchange chromatography (Dowex 1x8–200)
and measured as previously reported (18).
Analysis of water-soluble PtdCho metabolites
Cells were scraped and extracted for 1 h at 4 C before
adding chloroform and water to a final ratio of
CHCl3/MeOH/H2O = 1:1:0.9. To separate labeled
glycerophosphocholine, phosphocholine and choline, the
aqueous-methanolic phase was chromatographed on Dowex 50-WH+ according
to a modification of the procedure described by Cook and Wakelam (17, 19).
Assays of PLD activity by phosphatidylethanol (PtdEtOH) production,
and diacylglycerol (DAG) and phosphatidic acid (PA) determinations
PLD activity was evaluated measuring PtdEtOH formation from
[3H]myristic acid labeled PtdCho, which is considered a
direct and specific assay for PLD (20). After washing as described
above, cells were incubated with 1% ethanol for 10 min, and sCT was
added for the times indicated.
Cells were scraped from the culture dishes in ice-cold methanol, and lipids were extracted by the method of Bligh and Dyer (21): the monophase was vortexed, 1 vol of chloroform, 1 vol of 1 M KCl and HCl to 1 N final concentration were added, and the resulting aqueous and organic phases separated by centrifugation for 15 min at 3,000 x g. The lower phase was washed with synthetic upper phase (22), dried down under a stream of N2, stored at -80 C, and analyzed by TLC. DAG was separated by chromatography on 20 x 20 cm silica gel 60 plates, activated at 110 C for 1 h before use. The solvent system was petroleum ether/diethyl ether/acetic acid (80:20:1, vol/vol) (23) (DAG RF = 0.15–0.20).
For the analysis of PA and [3H]PtdEtOH lipid extracts were applied, along with standards, to 20 x 10-cm silica gel 60 plates, and separated using the solvent system ethyl acetate/iso-octane/acetic acid/water (13:2:3:10, vol/vol) (PA RF = 0.30). [3H]PtdEtOH was identified by comigration with unlabeled PtdEtOH prepared according to Baldini et al. (22).
Detection of PKC
For immunoblotting, 90% confluent cells were incubated in
serum-free DMEM containing 0.2% BSA for 1 h, then treated with
sCT. After the indicated time periods, the medium was removed, and
cells were washed twice with ice-cold PBS. Cells were then scraped into
1 ml of ice-cold homogenization buffer [20 mM Tris-HCl, pH
7.5, 1 mM EDTA, 1 mM EGTA, 2 mM
dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 10
mM benzamidine, 25 µg/ml leupeptin and 6 µg/ml
aprotinin]. All subsequent steps were carried out at 4 C. The cells
were lysed by ultrasonication and spun for 1 h at 100,000 x
g. Supernatants were used as a source of cytosolic protein.
Pellets were resonicated in 1 ml of the same buffer containing 1%
Triton X-100 and centrifuged for 1 h at 100,000 x
g, yielding the solubilized membrane fraction. The cell
fractions were subjected to SDS-PAGE (8% acrylamide gel), and proteins
were transferred onto nitrocellulose membranes. Filters were then
incubated at 4 C overnight with monoclonal antibodies against PKC,
, and isoenzymes (diluted 1:1000) and then with antimouse
horseradish peroxidase-conjugated secondary antibody (diluted 1:5000),
which was detected by ECL.
Cellular distribution of PKC isoenzymes was evaluated by standard indirect immunofluorescence.
Statistics
Data are presented as average ± SE.
Statistical analysis was performed by ANOVA. P < 0.05
was conventionally considered to indicate statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This was in agreement with what we
have previously reported (11).
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) but failed to raise the levels of
[3H]phosphocholine and
[3H]glycerophosphocholine measured in parallel and within
the same cells (data not shown). These results suggest that only
PtdCho-PLD was activated by sCT treatment, whereas neither PtdCho-PLC
nor PLA activity were affected (19). The increase of PLD-dependent
extracellular free [3H]choline was time and concentration
dependent (Figs. 1B and 2A). The effect
started above 10-9 M and reached a plateau at
10-6 M sCT, with an EC50 =
2.5 ± 0.5 x 10-8 M (mean ±
SE, n = 3, Fig. 1B).
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, the hormone induced a time-dependent increase of PA
generation, with a maximum (2-fold) achieved within 5 min, followed by
a sharp decrease probably due to conversion to DAG (see below).
Activation of PLD
In the presence of EtOH, activation of PLD can be measured in
[3H]myristic acid-labeled cells by monitoring the
formation of [3H]PtdEtOH. This is a specific and stable
product of the characteristic PLD-dependent transphosphatidylation of
PtdCho in the presence of a primary alcohol (24). 10-6
M sCT induced a time-dependent accumulation of
[3H]-PtdETOH, which was detectable within a few seconds
and reached a plateau in 5 min (Fig. 3).
These results are consistent with those observed for free
[3H]choline and PA.
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, the hormone induced a rapid DAG
increase occurring within a few seconds, followed by a slower
accumulation, reaching a maximum after 5 min, and subsequently
declining. The existence of this biphasic sCT response is consistent
with a different origin of the two DAG phases, as we have already
reported for vasopressin in myogenic cells (16). To confirm the PLD
origin of one of the two phases, before stimulation with sCT, cells
were pretreated for 15 min with 100 µM propranolol, a
specific PA-phosphatase inhibitor (25). Under these conditions,
analysis of DAG (Fig. 5) showed that the
late phase of sCT-dependent DAG generation, measured after 5 min of
incubation, was completely abolished by this agent, suggesting that
this component of DAG increase is entirely due to the conversion of PA
into DAG. In contrast, the first phase of DAG generation was not
inhibited by propranolol, suggesting that its origin is from PLC
activity (Fig. 5). It should be noted that pretreatment with
propranolol increased PLC-dependent DAG generation. This effect was
probably due to stimulation of PLC activity by the accumulated PA as we
and others have previously reported (16, 26). Similar results were
obtained with alprenolol (cpm, basal: 2858 ± 85;
10-6 M sCT, 5 min: 4236 ± 124; 100
µM alprenolol + 10-6 M sCT, 5
min: 2728 ± 185; mean ± SE, n = 3
P < 0.02).
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and 7
show analysis of the Ca2+/DAG-dependent PKC, the
DAG-dependent, Ca2+-independent PKC, and the atypical
PKC isoenzymes in cells treated with 10-6 M
sCT for the indicated time periods. We observed that while PKC and
were totally present in the soluble fraction in untreated cells,
the -isoenzyme was partially membrane linked, indicating a potential
constitutive activation of this enzyme. In cells treated with sCT, we
observed a time-dependent membrane translocation of the PKC isoform.
Potential activation of this isoenzyme was indicated by a similar
translocation induced by 10-7 M phorbol
13-myristate 12-acetate (PMA), a phorbol ester that is a potent PKC
activator in short-term treatments (time 0, cytosol 94 ± 4%,
membrane 6 ± 4%; 5 min PMA, cytosol 29 ± 6%, membrane
71 ± 6%, n = 3). However, sCT-dependent PKC membrane
translocation had a slower pattern compared with PMA because lower
levels of translocation were observed after 5 min with sCT (11 ±
6%, n = 3) relative to PMA (66 ± 10%, n = 3,
P < 0.01). Our data are consistent with a sustained
stimulation of PKC via PLD activation. After sCT treatment, PKC
showed a modest (19 ± 9%) but clearly detectable increase in the
particulate fraction, correlated with a simultaneous and comparable
decrease in the cytosol (Fig. 6A and 7). sCT did not induce any
significant effect on the PKC isoform (Fig. 6A and 7). This was not
due to compartmentalization of this isoform which, similar to the
and the isoenzymes, was found by immunofluorescence to be diffusely
distributed both in the nucleus and in the cytoplasm of unstimulated
LLC-PK1 cells (data not shown).
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,
, and membrane translocation, in the human BIN-67 ovarian
carcinoma cell line expressing the CTR48(+) isoform (Table 1). In contrast to results with the
LLC-PK1 cells, treatment of BIN-67 cells with either salmon
or human CTs failed to activate PLD (Table 1) or induce membrane
translocation of the PKC isoenzymes , , and (Fig. 6B). In the
same cells, parallel experiments showed that the PLD pathway was
activated by 10-7 M PMA (Fig. 3, inset), and that a PLC-coupled receptor agonist, bradykinin
(10-6 M), stimulated by 2-fold both InsP
formation and [Ca2+]i increase (not shown),
thus indicating that normal intracellular signals were operative.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and possibly isoenzymes. The capacity of
CTR to couple to multiple signal transduction pathways, including the
PLD, could provide an explanation for the diverse biological activities
of CT. CT-induced activation of adenylyl cyclase was the first
signaling mechanism recognized for the CTR, and activation of this
pathway is, for example, responsible for regulation of specific cell
functions, such as stimulation of the sodium pump (13), or arrest of
cell motility (27, 28, 29). It recently became apparent that not all of the
CTR-mediated cell functions can be explained by effects on increased
cAMP levels. For example, stimulation of Ca2+ signaling has
been shown to be responsible for osteoclast retraction, which, together
with the arrest of cell motility by cAMP, impairs bone resorption (29, 30). The predicted structure of the LLC-PK1 cell CTR exhibits several unique features that characterize this subfamily of receptors, including a short cytosolic loop that is not similar to corresponding regions (between helices V and VI) of other adenylyl cyclase-coupled receptors. This region, as well as the proximal portion of the carboxy-terminal end, does contain a potential consensus sequence for G protein coupling. In preliminary studies, we have used a chimeric IGF II/CTR receptor to demonstrate that these nucleotide sequences do encode amino acid cassettes that are able to interact with distinct G proteins (31, Orcel et al. manuscript in preparation). Thus, these unusual structural features may account for coupling of the CTR to different G proteins and for stimulation of multiple signal transduction pathways.
Phospholipase-dependent breakdown of phospholipids represents one of
the major contributors to hormone-mediated cell regulation. CT-mediated
activation of PtdIns-PLC leading to inositol trisphosphate-induced
Ca2+ mobilization has been demonstrated in
LLC-PK1 cells and in cells transfected with the porcine CTR
(11, 12, 32). The ultimate product of PtdIns metabolism is DAG, a
membrane lipid that binds DAG-dependent PKC, activating the catalytic
domain of the enzyme. DAG formation, however, is not an unique feature
of PtdIns turnover because it may also be derived from the metabolism
of PtdCho (24). This is a substrate for several phospholipases and in
this study we report that its metabolism is stimulated in
LLC-PK1 cells by treatment with sCT in a dose- and
time-dependent manner. Analysis of specific products of PtdCho
breakdown suggested that CTR activates PLD rather than PtdCho
specific-PLC or PLA. This is further confirmed by ethanol
transphosphatidylation, an event dependent upon specific PLD activation
(24). sCT-induced PLD activation markedly increased PA, a molecule that
has been implicated in signal transduction pathways related to the
induction of mitogenesis (33), for example by activating MAP kinase
(34), possibly explaining the cell cycle-dependent effect of sCT on
LLC-PK1 cells (13, 14). In addition, it has recently been
reported that PA directly activates PKC, an event independent of
both Ca2+ and DAG (22). Here we report a modest PKC
translocation, which could be due to sCT-induced PLD activation, and
subsequent PA formation.
Our results also demonstrate that DAG production is regulated with a
distinct temporal pattern, whether derived by PtdIns or PtdCho
metabolism. Examination of the time-dependent effects of sCT, and
the inhibition of sustained DAG production by propranolol (25),
indicate that activation of PtdIns-PLC is likely to induce a rapid,
transient increase in DAG formation, which is over within a few
seconds, whereas PLD activation stimulates a second, sustained
increment of DAG production lasting several minutes. This pattern may
explain the prolonged activation of the PKC isoenzyme observed in
sCT-treated cells. PKC is a classic Ca2+/DAG-dependent
PKC isoform, which is highly expressed in LLC-PK1 cells. CT
induces a slow and sustained PKC membrane translocation. This is at
variance with the pattern observed in cells treated with PMA, which
induced a rapid activation of the isoenzyme. These results suggest a
possible role played by PLD in the sCT-induced sustained PKC
activation because PLD-dependent DAG generation is quantitatively
predominant and generated for a prolonged time compared with the DAG
produced by PLC activation. Among the many biological roles for PKC,
the enzyme has been found to be involved in the modulation of the CTR
function and response to the hormone (35, 36, 37). Our data provide
insights indicating that PKC and are potential components of the
CTR(48-)-coupled signaling. In contrast, despite the generation of DAG
induced by sCT, translocation of the DAG-dependent PKC was not
detected. To explain this phenomenon, we examined the cellular
distribution of this isoenzyme by immunofluorescence but failed to
detect any compartmentalization that could prevent its activation by
surface receptor-coupled signals. Our results, however, were similar to
those observed with angiotensin II, thrombin, and bradykinin receptors,
which are known to generate DAG and translocate several PKC isoforms,
including and , but fail to translocate PKC in kidney
proximal tubule cells, platelets, and fibroblasts, respectively
(38, 39, 40).
The porcine CTR has a predicted structure that substantially differs
from the structure of the human CTR cloned from the ovarian carcinoma
cell line, BIN-67 (7). The latter shows higher affinity for sCT
relative to the porcine CTR. Although the two receptors are 75%
identical, comparison of the predicted structures reveals a substantial
difference, where the ovarian CTR contains a 48-bp cassette encoding a
16-amino acid insert located between the predicted first and second
transmembrane domains. Recent reports have demonstrated that this
insert greatly modifies the signaling properties of the receptor. In
fact, low cAMP production has been demonstrated in response to CT, and
PtdIns metabolism mediated by PLC is impaired in cells expressing the
ovarian CTR receptor, with consequent block of Ca2+
mobilization (7). In our hands, treatment of BIN-67 cells with either
sCT or human CT failed to activate PLA-, PLC-, and PLD-induced PtdCho
signals, as well as PKC activation of representatives of the classic
(), novel (), and atypical () PKC isoenzymes. We cannot
exclude that, similar to what observed for the PTH/PTHrp receptor (41, 42), the different responses to CT depend on cell type. However, in
this study we demonstrated that PtdIns metabolism and PLD signaling
were operative in the BIN-67 cells, as indicated by the
bradykinin-dependent stimulation of InsP formation and
[Ca2+]i increase, and by the PMA-induced
stimulation of PtdEtOH production, ruling out impairment of these
pathways as mechanisms preventing PtdIns and Pthd-Cho metabolism in
response to the hormone. Several lines of evidence indicate that the
48-bp cassette plays an inhibitory role in the signal transduction
mechanism coupled to CTR. For example, in transfection experiments
using the BHK-570 cell line, the CTR(48-), but not the CTR(48+),
isoform induce Ca2+ signaling (9). Similarly, preliminary
data obtained in our laboratory using 48(+) and 48(-) CTRs transiently
transfected in COS-7 cells confirmed that the 48(+) isoform shows a
response to CT strongly attenuated relative to the 48(-) isoform (43).
Taken together, these observations provide a reasonable evidence that
the lack of activation of intracellular signals is a property of the
CTR(48+) and not a cell-type specific phenomenon. Analysis of the
predicted amino acid sequence of the first intracellular domain,
including the 16-amino acid insert, reveals that this domain lacks the
consensus site for G protein binding and would, therefore, be predicted
not to directly couple to these signaling molecules (7). The 16 amino
acid insert may also modify the spatial conformation of the CTR that,
although permitting binding of the hormone to the receptor, prevents
its association with the phospholipase-coupled signal transduction
pathways.
In conclusion, we have demonstrated that the porcine CTR(48-) is
coupled to novel PLD-signal transduction events leading to sustained
DAG production and PKC and possibly activation in
LLC-PK1 cells. Further studies are necessary to define the
downstream events associated with the activation of these pathways. The
failure of the CTR48(+) isoform to activate the phospholipase dependent
pathways provides a novel mechanism by which structurally distinct CTR
isoforms could produce specific cell responses by selective activation
of different signals. Understanding of the molecular basis of this
phenomenon may help to establish treatment strategies that will permit
more effective use of CT.
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Acknowledgments |
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Footnotes |
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Received December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation is mimicked
by exogenous phospholipase D. Biochem J 321:497–501
, 
, and . Eur
J Biochem 225:297–304[Medline]
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