From the G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genova, Italy, ¶ Department of Experimental Medicine,
Section of Biochemistry, University of Genova, Viale
Benedetto XV 1, 16132 Genova, Italy,
Institute of Cybernetics
and Biophysics, CNR, Via De Marini 6, 16149 Genova, Italy and
** Department of Experimental Oncology, Istituto Nazionale per lo Studio
e la Cura dei Tumori, Via Venezian 1, 20133 Milano, Italy
Received for publication, November 21, 2001, and in revised form, March 6, 2001
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ABSTRACT |
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CD38 is a bifunctional ectoenzyme synthesizing
from NAD+ (ADP-ribosyl cyclase) and degrading
(hydrolase) cyclic ADP-ribose (cADPR), a powerful universal calcium
mobilizer from intracellular stores. Recently, hexameric connexin 43 (Cx43) hemichannels have been shown to release cytosolic
NAD+ from isolated murine fibroblasts (Bruzzone, S., Guida,
L., Zocchi, E., Franco, L. and De Flora, A. (2001) FASEB J. 15, 10-12), making this dinucleotide available to the ectocellular
active site of CD38. Here we investigated transwell co-cultures of
CD38+ (transfected) and CD38 CD38, a type II transmembrane glycoprotein of 46 kDa, formerly
known as a leukocyte activation antigen (1, 2), has attracted increasing attention since it proved to be a bifunctional ectoenzyme involved in the metabolism of two signal molecules, i.e.
cyclic ADP-ribose (cADPR)1
and NAADP+ (3, 4). CD38 is able either to convert
NAD+ to cADPR (ADP-ribosyl cyclase) and then to hydrolyze
cADPR (cADPR hydrolase), or to catalyze a base exchange reaction
leading to NAADP+ biosynthesis from NADP+
and nicotinic acid (3, 4). Cyclase and base exchange activities are
common to other members of the CD38 family, the best known of which is
a soluble protein purified and characterized from the marine mollusk
Aplysia californica (5, 6).
Since both cADPR and NAADP+ are potent calcium mobilizers
from distinct intracellular stores (3, 4), CD38 is held to play an essential role in the control of calcium homeostasis in many responsive cells. Specifically, cADPR has been demonstrated to regulate a number
of calcium-related cellular events including proliferation, contraction, and secretion (3). Therefore, cADPR can reach its receptor-operated intracellular stores (7), although its site of
CD38-catalyzed generation is in fact ectocellular (8, 9). This topological paradox holds both for CD38 in the plasma membrane and for
the subcellular fraction of CD38 whose active site is hidden inside
either exocytotic or endocytotic vesicles during intracellular
trafficking. Indeed, both enhanced exocytosis and ligand-induced
endocytosis of CD38-containing membrane vesicles proved to elude such
compartmentation and to be causally associated to
cADPR-dependent [Ca2+]i increases
(10, 11). Elucidation of the topological paradox of the CD38/cADPR
system came from some recent findings as follows. (i) The plasma
membrane of several cell types harbors a passive transport system for
pyridine dinucleotides, which is responsible for NAD+
fluxes through the membrane (11), thus providing NAD+
substrate to the otherwise inaccessible active site of CD38. This
dinucleotide transporter has been identified with connexin 43 hemichannels (12). (ii) Transmembrane CD38 is an active transporter of
catalytically produced cADPR across its oligomeric structure (13).
(iii) A third, CD38-unrelated mechanism of permeation of extracellular
cADPR across cell membranes has been postulated in selected cell types
(14, 15).
The presence of multiple transport systems for NAD+ and
cADPR in the plasma membrane raises the possibility of a paracrine exchange of these molecules between neighboring cells via Cx43, CD38,
and eventually cADPR influx. The possibility of
NAD+/cADPR-related paracrine mechanisms and their potential
role in regulating intracellular calcium were experimentally addressed in the present study by means of co-cultures of CD38 sense- and antisense-transduced 3T3 fibroblasts. CD38 Materials--
[32P]NAD+ (200 Ci/mmol)
and [3H]NAD+ (40 Ci/mmol) were obtained by
ICN (Milan, Italy) and PerkinElmer Life Sciences, respectively. cADPR,
[32P]cADPR, and [3H]cADPR were prepared
enzymatically from NAD+,
[32P]NAD+, and
[3H]NAD+, respectively, with recombinant
ADP-ribosyl cyclase from A. californica (courtesy of Prof.
H. C. Lee) and HPLC-purified (14). Cx43 antisense (5'-CTCCAGTCACCCATGTCTG-3') oligodeoxynucleotide, complementary to the
AUG translation start codon region of murine Cx43 mRNA and the
corresponding sense (5'- CAGACATGGGTGACTGGAG-3'), were purchased from
Life Technologies, Inc. Fura 2-AM was obtained from Calbiochem. The
anti-cADPR polyclonal antibody (16) and recombinant CD38 (17) were
kindly provided by Prof. H. C. Lee. All other chemicals were obtained
from Sigma.
Cell Lines--
NIH 3T3 cells obtained from ATCC (Manassas, VA)
were cultured as described (10). Transfection with sense
(CD38+) or antisense (CD38 Assay of Ectoenzyme Activities--
NAD+
glycohydrolase (NAD+ase), GDP-ribosyl cyclase, and cADPR
hydrolase activities were assayed on 1 mM NAD+,
1 mM nicolinamide guanine dinucleotide, and 0.5 mM cADPR, respectively, by incubating intact
CD38+/
CD38+-transfected 3T3 cells, but not the CD38 cADPR Influx in Intact CD38
Total membranes (5 mg/ml) and intact 3T3 CD38
HPLC analyses were performed on a Hewlett-Packard 1090 instrument
equipped with an HP1040 A diode array spectrophotometric detector set
at 260 nm, using a 5-µm, 100-Å, 150 × 3.9-mm Delta pack C18
reverse phase column (Waters, Milford, MA). Solvent A was water, and
solvent B was 70% 0.1 M KH2PO4
containing 5 mM PIC A reagent (Millipore, Milan, Italy), pH
5, and 30% methanol; the solvent program was a gradient starting at
100% solvent A for 5 min, linearly increasing to 10% solvent B in 25 min, and then increasing to 100% solvent B in 30 min at a flow rate of 0.5 ml/min. Identification and quantitation of the individual peaks
were obtained both by co-elution with known standard compounds and by
comparison of UV absorption spectra with those of computer-stored standards. cADPR eluted at 35 min, completely separated from other nucleotides and nucleosides. Sensitivity of this HPLC analysis of cADPR
was Co-culture Conditions--
CD38-antisense-transfected 3T3 cells
(0.25 × 106) were seeded in transwell plates (24 mm
diameter) on pre-established feeder layers of CD38+ or
CD38
Cx43 antisense and sense oligodeoxynucleotides were administered, each
at 20 µM, in phosphatidylcholine liposomes (12) to CD38+/ Fluorimetric Determination of
[Ca2+]i--
CD38
For determination of [Ca2+]i in
CD38 Determination of Intracellular cADPR in Co-culture
Conditions--
After 48 h co-culture on 75-mm diameter plates,
CD38 target cells were washed with 10 ml of PBS, detached with trypsin,
and washed twice with 1 ml of ice-cold PBS at 5,000 × g for 30 s. Pellets were resuspended in 250 µl of
cold water and frozen at Determination of Extracellular cADPR--
At various times of
co-culture of CD38 Determination of Extracellular NAD+--
Fresh
co-culture media at different times and in different experimental
conditions were collected and cells were removed by 3 subsequent
centrifugations at 5,000 × g for 30 s.
NAD+ content was measured on 200 µl aliquots by a
sensitive enzymatic cycling procedure (22), as described (11). To
calculate the percentage of cell lysis, hexokinase activity was assayed
in aliquots of the same media (11).
Cell Proliferation Assay--
CD38 Cell Cycle Analysis--
Following co-culture of
CD38 Susceptibility of CD38
Therefore, we investigated the possible permeation of extracellular
cADPR across the plasma membrane of native CD38
The data illustrated in Fig. 1 and the intrinsic occurrence of cADPR
receptors inside the 3T3 cells (10) prompted us to investigate whether
these intact cells respond to externally added cADPR with mobilization
of [Ca2+]i levels. As shown in Fig.
2, addition of the cyclic nucleotide to
CD38
These data demonstrate responsiveness of 3T3 fibroblasts to
extracellular cADPR, similarly to earlier results obtained on the same
permeabilized cells (10), yet with distinctive patterns of time
dependence, i.e. a fast peak of
[Ca2+]i elevation in the permeabilized cells (10)
and a sustained response in the native 3T3 fibroblasts (Fig. 2). The
latter pattern suggests influx of cADPR (and of both cADPR antagonists
as well) across the plasma membrane. Cx43 hemichannels are not involved in the [Ca2+]i increases elicited by
extracellular cADPR, in agreement with complete lack of cADPR transport
into proteoliposomes reconstituted with homogeneous Cx43 (12).
Effect of the Co-culture Over CD38+/
In an attempt to investigate whether a quantitative relationship exists
between the functional effects on the [Ca2+]i and
the extracellular concentration of NAD+ and cADPR, both
nucleotides were measured in the media of the various cultures shown in
Fig. 3 at different times. Results are reported in Table
I. Levels of extracellular
NAD+ proved to be remarkably stable throughout the
co-culture conditions up to 72 h of incubation. Moreover, they
were not significantly modified in the
CD38+/CD38
On the contrary, concentrations of extracellular cADPR, measured by
means of a specific RIA (16), were progressively increasing in the
CD38+/CD38
The results shown in Fig. 3 and in Table I demonstrate a good
quantitative correlation between [Ca2+] increases of 3T3
cells and the concentrations of extracellular cADPR in the
corresponding culture media. As mentioned, no such correlation was
observed with the levels of extracellular NAD+, which were
almost unaffected even under experimental conditions that result in the
enzymatic conversion of the dinucleotide (by either CD38, ADP-ribosyl
cyclase, or NAD+ase). The levels of extracellular
NAD+ reported in Table I seem in fact to reflect
steady-state concentrations arising from a two-step process of release
and enzymatic conversion (see "Discussion"). In any case, the
results shown in Fig. 3 suggest the involvement of extracellular
NAD+ and cADPR in the co-culture medium as responsible for
calcium mobilization in the CD38
In order to correlate better extracellular cADPR to intracellular
calcium release, the specific cADPR antagonists 8-NH2-cADPR and 8-Br-cADPR were separately added to the co-culture medium. As shown
in Fig. 4, supplementation of
extracellular 8-NH2-cADPR or of known membrane permeant
(26) 8-Br-cADPR (both at 1 µM) for 24 h completely
inhibited the [Ca2+]i increase in the
CD38 Role of Cx43 Hemichannels in the [Ca2+]i
Changes Observed in Mixed CD38+/CD38 Role of cADPR in the Changes Observed in Mixed CD38+/ Effect of the Co-culture Over CD38+/
Involvement of both extracellular NAD+ and cADPR in the
growth-enhancing effect was demonstrated by the significantly
(p < 0.05) higher rate of proliferation that was
observed upon co-culturing CD38
Therefore, the NAD+/cADPR-mediated paracrine cross-talk
leading to increases of [Ca2+]i in
CD38 Cell Cycle Analysis--
These experiments were performed in
co-culture conditions different from those followed for cell
proliferation assays (see "Experimental Procedures"). Specifically,
a larger culture surface (plates of 75 mm diameter) and an incubation
for 72 h were chosen in order to achieve a number of
CD38
In Table II the results of four different
BrdUrd pulse labeling experiments are reported. In CD38 The present investigation was focused on a simple, yet
informative, model of paracrine communication impacting on cell growth. Mixed co-culture of two populations of 3T3 fibroblasts differing from
each other for CD38 expression on their plasma membrane and use of
transwell systems avoiding contact between the two cell populations
allowed us to address the occurrence of a
NAD+/cADPR-related paracrine cross-talk. In addition, use
of selected reagents designed to modulate the corresponding effects in
the target CD38 Fig. 6 depicts the conclusions of this
study. Cx43 hemichannels mediate the release of NAD+ from
feeder cells, thus making it available to the ectocellular active site
of CD38 in their plasma membrane (12). Subsequent cADPR generation is
followed by its channeling across oligomeric CD38 to reach the cytosol
of the feeder cell, thus completing an autocrine loop (13), and also by
appearance of cADPR in the extracellular medium. The third step is
permeation of cADPR across the plasma membrane of target
CD38 3T3 cells in
order to establish the role of extracellular NAD+ and cADPR
on [Ca2+]i levels and on proliferation of the
CD38
target cells. CD38+, but not
CD38
, feeder cells induced a
[Ca2+]i increase in the CD38
target
cells which was comparable to that observed with extracellular cADPR
alone and inhibitable by NAD+-glycohydrolase or by the
cADPR antagonist 8-NH2-cADPR. Addition of recombinant
ADP-ribosyl cyclase to the medium of CD38
feeders induced
sustained [Ca2+]i increases in CD38
target cells. Co-culture on CD38+ feeders enhanced the
proliferation of CD38
target cells over control values
and significantly shortened the S phase of cell cycle. These results
demonstrate a paracrine process based on Cx43-mediated release of
NAD+, its CD38-catalyzed conversion to extracellular cADPR,
and influx of this nucleotide into responsive cells to increase
[Ca2+]i and stimulate cell proliferation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3T3 cells were
found to respond to the paracrine production of cADPR by co-cultured
CD38+ 3T3 cells with a calcium-related increase of
proliferation. This previously unrecognized interplay between
extracellular NAD+ and cADPR may represent a means for
regulating intracellular calcium homeostasis and relevant cell
responses in selected tissue microenvironments
featuring CD38+ stromal cells and CD38
parenchymal cells, e.g. bone marrow (14) and smooth muscle (15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) CD38 cDNA was
performed as described (10). Transfected cells were routinely
maintained under geneticin (1 mg/ml) selection.
3T3 cells (106) in 400 µl of PBS
containing 10 mM glucose (PBS/glucose) at 37 °C. At
different times, 60-µl aliquots of the incubation mixtures were
centrifuged for 30 s at 5,000 × g, and the
corresponding supernatants were deproteinized with trichloroacetic acid
(10% final concentration) as described (14). HPLC analyses of
nucleotides in the samples were performed as described (15). Protein
content was determined according to Bradford (18).
ones, expressed at their outer surface the three enzymatic activities
of CD38 as follows: NAD+ase (29 ± 3 nmol of
ADPR/min/mg), GDP-ribosyl cyclase (2.8 ± 0.2 nmol of
cGDPR/min/mg), and cADPR hydrolase (0.61 ± 0.04 nmol of ADPR/min/mg). Nicolinamide guanine dinucleotide was used as
substrate for the cyclase activity because its enzymatic product cGDPR
is not hydrolyzable and accumulates during the assay (19).
3T3 Cells and cADPR
Association to Their Membranes--
Cell membranes were prepared by
submitting CD38
3T3 cells (50 × 106) to lysis in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3 M sucrose, in the presence of
protease inhibitors (10 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml trypsin inhibitor) at 0 °C. Following sonication in ice for
1 min at 3 watts (Heat-System Ultrasonics, W-380, New York), cell
lysates were subjected to two subsequent centrifugations at 4 °C as
follows: at 3,000 × g for 10 min and the corresponding
supernatants at 100,000 × g for 15 min. Membrane pellets were then washed twice in 2 ml of PBS buffer.
cells
(4 × 106/ml) were incubated in 1.0 ml of PBS/glucose
in the presence of 200 µM cADPR at 37 °C. At different
times 100-µl aliquots were withdrawn and centrifuged at 100,000 × g for 15 min (total membranes) or at 5,000 × g for 30 s (intact cells). Pellets were washed in 2 ml
of PBS/glucose, in order to dilute cADPR in the supernatants to
HPLC-undetectable concentrations, and then were resuspended in 300 µl
of water and sonicated for 1 min at 3 watts in ice. Aliquots of 270 µl were trichloroacetic acid-deproteinized (14), and cADPR content
was analyzed by HPLC (see below). Protein content was determined on
30-µl aliquots according to Bradford (18).
10 pmol per sample.
3T3 cells (1.0 × 106), in 1.5 ml
of fresh complete medium. Co-cultures were exposed to different cADPR
antagonists (20) as follows: 1 µM 8-Br-cADPR for 24 h or 8-NH2-cADPR for 24, 48, and 72 h; or to the gap
junction inhibitor oleamide (50 µM) for 24 h (21);
or incubated with Cx43 sense or antisense oligodeoxynucleotide (12) as
described below. Purified NAD+ase (Sigma) was added to 3T3
CD38+/
co-cultures at a final concentration of 1.3 milliunits/ml. Recombinant ADP-ribosyl cyclase from A. californica was added to 3T3 CD38
/
co-cultures at a final activity of 3 nmol of cADPR/min/ml. Parallel long term incubation of ADP-ribosyl cyclase with 100 µM
NAD+ revealed the formation of products other than cADPR,
among which are ADPR, ADP, and AMP.
feeders as well as to CD38
target
cells pre-adsorbed on a 20-mm diameter coverslip, separately. After
16 h of culture at 37 °C, cells were refed with complete Dulbecco's modified Eagle's medium, and
CD38
-treated cells were transferred onto transwell plates
over CD38+/
-treated feeders.
[Ca2+]i of CD38
3T3 target cells
was determined after 24 h of co-culture.
3T3 target cells
(2.5-5 × 104) adherent on 20-mm diameter coverslips
treated with or without Cx43 oligodeoxynucleotides were incubated in
the presence of Fura2-AM (10 µM) for 45 min at 37 °C.
Untreated cells on the coverslips were incubated without or with 50 µM oleamide or 50 µM 8-Br-cADPR or 100 µM 8-NH2-cADPR in zero calcium standard
solution (10) in a 200-µl recording chamber mounted on the stage of
an inverted microscope (Zeiss IM35, Stuttgart, Germany). After 20 min
incubation at 25 °C, 100 µM cADPR was added into the
chamber, and intracellular calcium concentration in Fura2-AM-treated cells was continuously recorded for 30 min, as described (11). No
differences were observed between CD38 antisense-transfected and
native CD38
3T3 cells as concerns the
[Ca2+]i changes.
cells co-cultured with CD38+/
feeders,
target CD38
cells were harvested at different times from
the transwells, washed twice in 1 ml of PBS for 30 s at 5,000 × g, and resuspended in 1 ml of fresh complete medium.
Calcium measures were performed in a 2-ml cuvette under continuous
stirring in zero calcium solution, as described (14). Statistical
analysis of different [Ca2+] values was performed using
one-way analysis of variance and two-sided Dunnett's t
test. p values were considered statistically significant
when <0.05.
20 °C and then thawed and sonicated in
ice 1 min at 3 watts. A 50-µl aliquot was withdrawn for assay of
protein (18), whereas the rest of the sample was deproteinized with
10% trichloroacetic acid (14). The cADPR content of the cell extracts
was analyzed by two subsequent HPLCs after addition of trace amounts of
radiolabeled [H3]cADPR (2 × 1,000 cpm) as internal
standard (14). Identification of the cADPR peak in the cell extracts
was confirmed by co-elution with the radioactive standard, by
comparison of the absorbance spectrum and elution time with standard
cADPR, and by the disappearance of the corresponding peak in the
matched CD38-hydrolyzed samples (14). The concentration of
intracellular cADPR was calculated from the area of the HPLC peak,
taking into account the percentage of nucleotide recovery obtained with
the radioactive standard.
over CD38+/
3T3 feeder
cells in complete medium (without phenol red), the medium was collected
and clarified by three repeated centrifugations at 300 × g for 5 min. The cell-free medium was trichloroacetic
acid-deproteinized (14) and submitted to enzyme digestion to hydrolyze
nucleotides potentially interfering with the cADPR assay (16). cADPR
content in the samples was determined by a sensitive and specific
radioimmunoassay (RIA) (16), rather than by HPLC as for intracellular
cADPR levels (see above), because high salt concentrations in these
samples proved to interfere with the latter type of analyses.
3T3target
cells were co-cultured in triplicate over CD38+/
feeders
as described above; after 24, 48, and 72 h, target cells were
harvested from the transwell with trypsin and washed twice in PBS at
5,000 × g for 30 s, and the dry cell pellet was
frozen at
20 °C. The DNA content of the cell pellets was estimated
with the CyQuant proliferation assay kit (Molecular Probes, OR). DNA fluorescence of the samples was measured on an LS-50B fluorometer (PerkinElmer Life Sciences). Results were expressed as percentages of
proliferation compared with control cells (CD38
3T3 cells
co-cultured with CD38
3T3 cells). The Mann-Whitney rank
sum test was used to determine the significance of the difference
between two cell populations.
cells (0.5 × 106) with
CD38+/
3T3 feeder cells (2 × 106) on
75-mm diameter transwell plates for 72 h, target cells were washed
in 10 ml of PBS and incubated for 30 min with 30 µM
5'-bromodeoxyuridine (BrdUrd). Cells were then washed with PBS and
either immediately fixed (zero time) or further co-cultured on
CD38+ or CD38
3T3 feeders for 4 h.
BrdUrd-labeled cells were detached with trypsin, washed twice in
ice-cold PBS containing 2 mM EDTA, and prepared for flow
cytometry-mediated analysis of BrdUrd and DNA contents as described
previously (10). Briefly, from flow cytometry-mediated and derived data
of the CD38
target cells cultured on either
CD38
or CD38+ feeder cells, the length of the
S phase (TS) was calculated on the basis of the fact
that BrdUrd-labeled cells were allowed to progress through the cell
cycle in a BrdUrd-free environment during the so-called
"post-labeling" period (4 h). Specifically, TS
values were calculated by comparing the mean DNA content of the cohort
of BrdUrd-labeled cells which have moved through the cell cycle during
post-labeling time, with that of G1 and G2/M
phase cells, using the relative movement (RM) method (23). RM values
were obtained using Equation 1,
where F is the mean red fluorescence of the
corresponding phase of the cell cycle. TS (the
length of the S phase) values were calculated from Equation 2,
(Eq. 1)
where RMT0 is relative movement at the
time of pulse labeling, RMT4h = relative
movement 4 h after pulse labeling, and 4 is the observation time
of 4 h. The Student's t test was used to determine the
statistical significance of the difference between the two cell
populations in the four experiments performed.
(Eq. 2)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3T3 Fibroblasts to
Extracellular cADPR--
Digitonin-permeabilized murine 3T3
fibroblasts have been shown previously to respond to cADPR with an
immediate, 8-NH2-cADPR-inhibitable elevation of
[Ca2+]i levels (10). Influx of external cADPR has
been postulated in cells as murine B-lymphocytes (24), rat cerebellar
granule neurons (25), and smooth myocytes from bovine trachea (15),
where sustained [Ca2+]i increases were observed
following exposure to extracellular cADPR. Moreover, cADPR influx was
measured directly in human hemopoietic progenitors, in which the
alternative possibility of surface-bound cyclic nucleotide was ruled
out by time dependence of cADPR association to extensively washed cells (14).
3T3
fibroblasts. Both intact cells and isolated membrane preparations were
examined for their content of HPLC-detectable cADPR at different times
of incubation with 200 µM cADPR (Fig.
1). While membrane-associated cADPR kept
stable over time at barely detectable levels, there was a progressive
increase of the fraction of cell-associated cADPR which resisted
washing of the incubated fibroblasts. This time dependence of
association to equally washed cells and failure to record association
to isolated cell membranes strongly favor a process of internalization
of external cADPR over a simple surface binding.
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Fig. 1.
Time-dependent influx of
extracellularly added cADPR into intact CD38
fibroblasts. Intact CD38
3T3 cells (
) or their
membranes (
), prepared as described under "Experimental
Procedures", were incubated in the presence of 200 µM
extracellular cADPR at 37 °C. At the times indicated, aliquots were
withdrawn, and the cADPR content of each sample was analyzed by HPLC as
described under "Experimental Procedures."
native cells resulted in a progressive increase of
their [Ca2+]i levels. The lowest effective
concentration of cADPR was 0.5 µM, similarly to results
obtained with bovine tracheal smooth myocytes (15). The kinetics and
extent of [Ca2+]i elevations were identical to
those recorded in the same 3T3 fibroblasts exposed to oleamide, a known
inhibitor of solute exchange across gap junctions (21) and of specific
NAD+ transport through Cx43 hemichannels in isolated 3T3
cells (12). Also, the progressive [Ca2+]i
increase was completely unaffected in CD38
cells
pre-treated either with a specific anti-Cx43 deoxynucleotide or with
the corresponding sense deoxynucleotide. Conversely,
[Ca2+]i levels in CD38
cells
pre-incubated with either of two cADPR analogs and antagonists (100 µM 8-NH2-cADPR or 50 µM
8-Br-cADPR) failed to increase following addition of extracellular
cADPR.
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Fig. 2.
Responsiveness of CD38 3T3
fibroblasts to extracellular cADPR. CD38
3T3 cells,
either untreated (control) or treated with various Cx43 modulators and
cADPR antagonists (see "Experimental Procedures"), were exposed to
extracellular 100 µM cADPR. [Ca2+]i
changes were continuously measured as described (11). Traces
are the mean of 5 different experiments; no S.D. values are shown for
the sake of clarity.
Feeders on
[Ca2+]i of CD38
3T3
Cells--
These results and the availability of both
CD38
and CD38+ 3T3 fibroblasts prompted us to
develop a model for co-culture where CD38+ fibroblasts were
used as feeders for CD38
cells. Control co-cultures were
grown on a feeder represented by the same CD38
cells. The
properties we investigated in the CD38
3T3 cells were the
[Ca2+]i levels and also the rate of cell
proliferation, which had been proved to be enhanced under conditions of
de novo expression of CD38 cells resulting in cADPR-mediated
increases of the [Ca2+]i (10). Co-culturing with
CD38+ 3T3 feeder cells determined a progressive increase of
the [Ca2+]i of CD38
fibroblasts
from a basal value of 20.3 ± 2 to 49.6 ± 4 nM
within 72 h (Fig. 3). No increase
whatsoever was detectable in the same cells grown in the same
conditions, yet over CD38
3T3 feeders. Addition of
NAD+ase to the culture medium substantially decreased
(p < 0.05) the sustained enhancement of
[Ca2+] of cells co-cultured with the CD38-transfected
fibroblasts (Fig. 3), indicating a causal role of extracellular
NAD+ on the [Ca2+]i changes.
Moreover, supplementation of soluble ADP-ribosyl cyclase to the medium
conditioned by the presence of CD38
feeder cells elicited
a significant (p < 0.05) and sustained increase of
[Ca2+]i in the CD38
3T3 fibroblasts
over the remarkably stable levels observed in the same cells without
any addition (Fig. 3). This increase witnesses NAD+ release
from the CD38
feeder cells.
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Fig. 3.
[Ca2+]i changes in
CD38 3T3 cells co-cultured with
CD38+/
feeders. Co-cultures
were performed as described under "Experimental Procedures." At the
times indicated CD38
target cells were harvested, and
[Ca2+]i was determined (10), and extracellular
NAD+ and cADPR levels were measured as reported under
"Experimental Procedures." CD38
feeders,
;
CD38+ feeders,
; CD38
feeders plus
recombinant ADP-ribosyl cyclase,
; CD38+ feeders plus
NAD+-glycohydrolase,
. Values are means ± S.D. of
10 different experiments.
co-cultures nor in the
CD38
cultures supplemented with ADP-ribosyl cyclase as
compared with their corresponding CD38
control cultures.
The only difference was observed in the CD38+ cultures
supplemented with NAD+ase, where extracellular
NAD+ was slightly (18%) lower than in CD38
cultures alone (Table I).
Extracellular NAD+ and cADPR levels in the media from
CD38+/CD38 co-cultures and from control cultures
0.5% as determined by release of
hexokinase activity (see "Experimental Procedures"). Accordingly,
the NAD+ and cADPR concentrations measured in the media were
not determined by cell lysis during the co-cultures.
co-cultures, until reaching
6.0 ± 0.8 nM at 72 h (Table I). They were
remarkably lower in the medium from the same co-cultures supplemented
with NAD+ase, in agreement with a comparatively reduced
[Ca2+]i increase (Fig. 3). Addition of
ADP-ribosyl cyclase to the CD38
feeders resulted in
increasing concentrations of extracellular cADPR, although lower than
those measured in CD38+/CD38
co-cultures.
Possible reasons for this apparent discrepancy are as follows: (i) the
ADP-ribosyl cyclase is partially inactivated at 37 °C (19); (ii) the
Km value of ADP-ribosyl cyclase for NAD+
is higher (39 µM) than that of CD38 (14 µM)
(19); (iii) long term incubation of ADP-ribosyl cyclase on
NAD+ resulted in generation of nucleotides other than cADPR
(see "Experimental Procedures").
target cells.
target cells grown over the CD38+ feeder
cells. These cADPR antagonists had no effect on
[Ca2+]i levels of target cells co-incubated over
CD38
feeder cells (not shown). Extracellularly added
8-NH2-cADPR has been recently found to inhibit the
[Ca2+]i increase elicited by cADPR in human
hemopoietic progenitors (14) and in tracheal smooth myocytes (15).
View larger version (15K):
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Fig. 4.
Effect of cADPR antagonists and Cx43
inhibitors on the [Ca2+]i increase in
CD38 3T3 cells co-cultured over CD38+
feeders. CD38
target cells were co-cultured for
24 h on CD38
feeder cells (column 1) or
CD38+ cells in the absence (column 2) or
presence of 1 µM 8-NH2-cADPR (column
3), or 1 µM 8-Br-cADPR (column 4), or 50 µM oleamide (column 5). CD38
target cells pre-treated with Cx43 antisense (column 6) or
sense (column 7) oligodeoxynucleotide were co-cultured
24 h on CD38+ feeder cells pre-treated with the same
oligodeoxynucleotide. [Ca2+]i measurements were
carried out as described (10). Values are means ± S.D. of five
different experiments.
Co-cultures--
The increases of [Ca2+]i
induced in the target CD38
3T3 fibroblasts by
co-culturing with CD38+ feeder cells demonstrate a
paracrine role of extracellular NAD+ and cADPR in the
mechanism underlying these changes. Specifically, NAD+
release from cells followed by CD38-catalyzed generation of
extracellular cADPR seem to be the required steps. Since
NAD+ release has now been shown to take place in isolated
3T3 cells across hexameric hemichannels of Cx43 (12), we attempted to
disrupt the paracrine effects of co-culture by inhibiting the
NAD+-exporting activity of Cx43 hemichannels. Oleamide
proved to block the [Ca2+]i increase in our
co-culture setting almost completely (Fig. 4). Moreover, the specific
anti-Cx43 oligodeoxynucleotide inhibited the
[Ca2+]i increase in the target CD38
cells, whereas the corresponding sense deoxynucleotide was totally uneffective (Fig. 4). These results give further support to the idea of
Cx43-mediated export of cellular NAD+ and of subsequent
generation of extracellular cADPR at the outer surface of the
CD38+ feeder cells followed by influx of cADPR into the
target CD38
cells across a Cx43-unrelated transport
system (Fig. 2).
Co-cultures--
In an effort to directly demonstrate this paracrine
mechanism, we measured intracellular cADPR in the target
CD38
cells during the co-culture experiments.
CD38
cells were incubated on 75-mm diameter transwell
plates over pre-established CD38
/+ feeder
layers for 48 h in the same conditions used for cell cycle analysis (see below). Cell extracts were then analyzed by HPLC. The
intracellular cADPR concentration was undetectable in the CD38
target cells co-cultured over homologous
CD38
layers (controls), whereas it was estimated to be
2.1 ± 0.1 picomoles/mg in the CD38
grown on
CD38+ feeders. This value is in the range of reported
intracellular concentrations of cADPR in constitutively
CD38+ human lymphoid and myeloid cell lines (27, 28).
Feeders on the
Proliferation of CD38
3T3 Cells--
De novo
expression of CD38 has been demonstrated to enhance the rate of
proliferation of some cell types, including 3T3, via increases of
[Ca2+]i elicited by intracellular cADPR (10). In
order to investigate whether the calcium mobilization in
CD38
cells which is induced by cADPR generated and
provided by CD38+ cell feeders could interfere with cell
growth, we assayed proliferation of CD38
3T3 target cells
co-cultured with CD38+/
3T3 feeder cells. A significant
(p < 0.05) increase in cell proliferation was observed
in CD38
fibroblasts co-cultured over CD38+
cells for 72 h as compared with the same CD38
cells
grown on homologous CD38
feeders (Fig.
5A). This increase was
inhibited by addition of NAD+ase to the medium, with a
maximum effect being recorded after 24 h of culture
(p < 0.05). The reduced extent of inhibition afforded by NAD+ase at 48 and 72 h, despite the appearance of
detectable levels of extracellular cADPR (Table I), might reflect some
compensatory mechanisms promoting cell growth and either located
downstream of [Ca2+]i levels or independent of
them.
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Fig. 5.
Modulation of cell growth of
CD38 3T3 cells on
CD38+/
feeders. A,
CD38
target cells were co-cultured on CD38+
feeders in the absence (white columns) or presence of
NAD+ase (black columns). B,
CD38
target cells were co-cultured on CD38
feeders in the absence (control) or presence of recombinant ADP-ribosyl
cyclase. Results of 10 different experiments are expressed as
percentages of proliferation compared with control cells.
/
cells yet
in the presence of recombinant ADP-ribosyl cyclase added to the medium
(Fig. 5B). As observed with NAD+ase
supplementation (Fig. 5A), also in these experiments there was a weak quantitative correlation between time-dependent
increases of [Ca2+]i (Fig. 3) and corresponding
stimulation of cell growth. Both apparent discrepancies of timing and
of extent of effects seem to reflect additional, probably
[Ca2+]i-unrelated mechanisms involved in the
control of cell proliferation, especially on a long time scale.
However, the growth-promoting role of extracellular cADPR was
demonstrated by complete abolition of the increases of proliferation
afforded by CD38+ feeders on CD38
target
cells that was observed at all times investigated upon adding
8-NH2-cADPR (1 µM) to the co-culture media
(not shown).
target cells proved to have an important functional
outcome, i.e. enhanced cell proliferation.
target cells sufficient for fluorimetric detection
of BrdUrd. These conditions resulted in a [cADPR]e
concentration of 18 ± 2.1 nM (not shown).
target cells co-cultured over CD38
feeder cells the RM
showed a low variability among the four experiments, and the
corresponding TS values ranged from 19 to 28 h
with a mean value of 23 ± 4.4 h. In CD38
target cells co-cultured with CD38+ feeder cells the RM
showed a wide variability, indicating a less uniform progression
through the cell cycle, and the corresponding TS
values ranged from 4 to 16 h with a mean value of 10 ± 5.4 h. Statistic significance of values obtained with the two cell
populations (p < 0.01) confirmed a shorter S phase for the CD38
target population over CD38+ feeders
as compared with the control one over CD38
cells.
DNA synthesis time (TS) of CD38 3T3 cells co-cultured
over CD38+/
feeders
target cells were labeled for 30 min with 30 µM BrdUrd. At the times indicated cells were
fixed, processed with anti-BrdUrd monoclonal antibody and propidium
iodide (DNA content). Measurements of TS were
performed from the relative movement (RM) as described under
"Experimental Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (NAD+ase, ADP-ribosyl
cyclase, 8NH2-cADPR, and 8-Br-cADPR) enabled us to dissect
the individual steps of this intercellular communication and to
identify specific roles of NAD+ and of cADPR therein.
cells (Fig. 1), as previously suggested to occur in
several cell types responding to extracellular cADPR with calcium
mobilization and in some cases with remarkable changes in cell
functions (14, 15, 24, 25).
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Fig. 6.
The
NAD+/cADPR/[Ca2+]i relationship and
its role in regulation of proliferation of
CD38 target 3T3 fibroblasts
co-cultured with CD38+ feeders. For details see
text.
The paracrine model summarized in Fig. 6 envisages new roles for NAD+ as a cell-to-cell communication signal mimicking a hormone, whereas extra/intracellular cADPR represents its second messenger, and intracellular calcium behaves as a third messenger regulating selected cell functions (3, 9, 29). Recently, a comparable yet intracellularly localized loop has been described in rat heart mitochondria, where opening of the permeability transition pore is followed by release of intramichondrial NAD+. This dinucleotide can accordingly behave as substrate for the NAD+-glycohydrolase located outside the matrix space (30) which has been shown to express ADP-ribosyl cyclase activity (31). Therefore, release of NAD+ through mitochondrial permeability transition pore is expected to produce cytosolic cADPR with consequent calcium release from the sarcoplasmic reticulum (30).
Although the paracrine process involving NAD+ and cADPR is
demonstrated by the present results, further studies are required to
elucidate completely the quantitative aspects of this novel mechanism
of cell-to-cell communication. A challenging point is represented by
extracellular levels of NAD+ and cADPR. Both represent
steady-state concentrations resulting from a three-step process,
i.e. efflux of NAD+ from feeder cells, its
ectocellular conversion to cADPR, and eventually permeation of the
cyclic nucleotide across the plasma membrane of target cells.
Therefore, stability of [NAD+]e in the co-culture
experiments where it is measurably converted to cADPR could be
explained by an enhanced, Cx43-mediated release from feeder cells
resulting from a continuous and steep gradient of NAD+
concentrations across their plasma membrane. This enhanced efflux seems
to mimic closely the experimental situation previously observed upon
submitting cultured CD38 fibroblasts to extensive and
repeated washings (11). Another, probably related issue is that the
ectocellular ADP-ribosyl cyclase activity of CD38 in the feeder cells
is apparently working at largely non-saturating concentrations of
NAD+ (Table I), since its reported Km is
14 µM NAD+ (19). Accordingly, generation of
cADPR in the extracellular space could play a limiting role in this
complex process. Finally, the next step, i.e. clearance of
extracellular cADPR by the CD38
target cells, requires
elucidation of the transport system responsible for cADPR influx (Fig.
1) whose molecular properties are as yet unknown.
With respect to this, an interesting feature is the remarkably high
efficiency in the co-culture system of extracellular cADPR concentrations as low as 4.0-6.0 nM (Table I) in
triggering [Ca2+]i increases that are comparable
in extent to those elicited by cADPR added at concentrations several
orders of magnitude higher. Indeed, pulse addition of extracellular
cADPR below 0.5 µM was totally uneffective on
[Ca2+]i levels of CD38 cells (not
shown). A closely comparable situation has been recently reported for
the potent hemopoietic inhibition mediated by interferon-
(IFN-
)
constitutively expressed in the stromal microenvironment of human bone
marrow cultures (32). In this case, similar decreases of early
hemopoietic progenitors were observed with 20 units/ml of endogenous
IFN-
as with exogenous concentration of 200 units/ml added every day
or with 1,000 units/ml added weekly (32). Reasons for this
concentration disparity of IFN-
(32) and of cADPR as well (this
study) are as yet undefined and might depend on the physical
presentation of both signal molecules to their target cells.
Specifically, cADPR concentrations in the co-culture media could be in
fact non-homogeneous and locally higher in proximity to the
CD38
cells. An alternative explanation might be provided
by additional compounds being involved in sensitizing this signaling
process; simultaneous release from feeder CD38+ cells of
other signal metabolites enhancing cADPR efficiency, e.g.
dimeric ADP-ribose (33), could make the difference with exogenously supplemented cADPR.
The enhanced cell proliferation induced by CD38+ feeders in
CD38 target cells was prevented by the presence of
8-NH2-cADPR in the medium. This demonstrates a direct and
causal relationship between cADPR generated extracellularly by the
CD38+ feeder, [Ca2+]i increase, and
enhanced proliferation of CD38
cells. As far as the
latter process is concerned, the extent of increase of cell
proliferation recorded in the BrdUrd experiments following 72 h of
co-culture of CD38
3T3 cells over CD38+
feeders, accounting for ~100% (Table II), does not match with the
50% increase observed after the same time of co-culture in the
experiments measuring total DNA content (Fig. 5A). This
discrepancy can be reasonably due to the different co-culture
conditions used in the two types of experiments and especially to the
larger culture surface (75-mm diameter plates) required to obtain a
number of CD38
target cells sufficient for fluorimetric
detection of BrdUrd. Specifically, the higher number of
CD38+ feeder cells obtained at the end of the 72-h
co-culture generated comparatively higher [cADPR]e, as
witnessed by a concentration of 18 ± 2.1 nM
versus 6.0 ± 0.8 nM (Table I). Finally,
the time-dependent increase of the proliferation rate
measured at 24, 48, and 72 h in CD38
target cells
co-cultured over CD38+ feeders (Fig. 5A), which
likely reflects a progressive shortening of the S phase of the cell
cycle until reaching a TS value of 10 ± 5.4 h at 72 h (Table II), parallels the increase of
[cADPR]e in the media (Table I).
The growth-enhancing effect featured by CD38+ cell feeders
on CD38 target fibroblasts by virtue of a paracrine
NAD+/cADPR mechanism may have important functional
consequences that should extend beyond our model system of co-culture.
For instance, in bovine tracheal strips, we were able to show that
co-incubation of mucosa CD38+ fragments with smooth
myocytes induces the NAD+/cADPR-mediated increase of
[Ca2+]i in these cells (15). Moreover, a
cADPR-dependent expansion of human hemopoietic progenitors
grown on CD38+ stroma cells has been recently observed in
our laboratory (34). The mechanism underlying the calcium-related
stimulation of cell growth proved to be a significant shortening of the
S phase of the cell cycle (Table II). A comparable change had been
observed in an earlier study exploring the biochemical consequences of de novo expression of CD38 obtained upon transfecting
constitutively CD38
cells, i.e. murine 3T3
fibroblasts and human HeLa cells (10). Therefore,
[Ca2+]i increases that follow either enhanced
intracellular traffic of NAD+ and cADPR (9) or an
extracellular exchange of both signal metabolites can trigger an
increased cell proliferation via a significant shortening of the S
phase of cell cycle.
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FOOTNOTES |
---|
* This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro, MURST-PRIN 2000, MURST-CNR (5% Project on Biotechnology to A. D. F.), and from the University of Genova and CNR (Target Project on Biotechnology to E. Z.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from Fondo Sociale Europeo-MURST.
To whom correspondence should be addressed: Department
of Experimental Medicine, Section of Biochemistry, University of
Genova, Viale Benedetto XV 1, 16132 Genova, Italy. Tel.:
39-010-3538155; Fax: 39-010-5221944; E-mail: toninodf@unige.it.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M010536200
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ABBREVIATIONS |
---|
The abbreviations used are:
cADPR, cyclic
ADP-ribose;
Cx43, connexin 43;
NAADP+, nicotinic acid
adenine dinucleotide phosphate;
cGDPR, cyclic GDP-ribose;
NAD+ase, NAD+-glycohydrolase;
HPLC, high
pressure liquid chromatography;
PBS, phosphate-buffered saline;
RIA, radioimmunoassay;
BrdUrd, bromodeoxyuridine;
IFN-, interferon-
.
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