1 Department of Morphology, University of Geneva Medical School, 1211 Geneva 4,
Switzerland
2 Department of Genetics and Microbiology, University of Geneva Medical School,
1211 Geneva 4, Switzerland
* Author for correspondence (e-mail: david.caton{at}medecine.unige.ch)
Accepted 25 February 2003
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Summary |
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Key words: Cx32, Cx36, Cx43, Gap junctions, Lentiviral vectors, Pancreatic ß-cells
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Introduction |
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It has not yet proved feasible to assess whether this selective versatility
also applies to acute changes in the Cx pattern of adult cells, mostly because
it is difficult to obtain rapid, stable transfection of a foreign cDNA in
primary cells without losing the expression of the native differentiation
genes, particularly if the target cells divide poorly
(Anderson et al., 2002;
Nielsen et al., 1989
). To
overcome these problems, we have taken advantage of novel lentiviral vectors
which sustain expression of foreign genes in primary, fully differentiated and
non-dividing cells (Gallichan et al.,
1998
; Ju et al.,
1998
; Naldini et al.,
1996a
; Naldini et al.,
1996b
; Salmon et al.,
2000
). Here, we show that these vectors can be designed to express
efficiently a range of Cx-encoding cDNAs and to change both the number of gap
junction plaques and the coupling extent of primary, fully differentiated and
non-dividing cells.
In these experiments, we have used as a model the insulin-producing
ß-cells of pancreatic islets. These endocrine micro-organs, which are
mostly made up of ß-cells, express Cx36
(Serre-Beinier et al., 2000)
but no Cx26 or Cx32, which are found in pancreatic acini
(Meda et al., 1993
). The
distribution of Cx43 is less clear: it is expressed in some pancreatic vessels
and fibroblasts (Theis et al.,
2001
) and possibly also by ß-cells, at least under certain
conditions (Collares-Buzato et al.,
2001
; Meda et al.,
1993
). This distribution suggests that specific Cx isoforms and
the selective coupling that they mediate are required for the proper
functioning of different pancreatic cell types. In particular, several lines
of evidence suggest that Cx-mediated communication between ß-cells is
required for control of insulin secretion. In rats, sequential changes of
pancreatic function were found to parallel changes in Cx36 expression
(Calabrese et al., 2001
).
Expression of Cx32 in ß-cells of transgenic mice reduced insulin
secretion in response to physiological concentrations of glucose, indicating
that the in vivo expression of a Cx, which is exogenous to pancreatic islets,
perturbed b-cell functioning, in spite of the persistence of the native Cx36
and increased cell-to-cell coupling
(Charollais et al., 2000
).
To assess whether the type and level of Cxs affect insulin secretion
differently, we tested the expression of distinct Cx cDNAs in primary
ß-cells. Because these experiments are not feasible using standard
transfection methods owing to the very low proliferation rate of fully
differentiated islet cells (Nielsen et
al., 1989), we transduced the cells with Cx-encoding lentiviral
vectors. For these experiments, we did not choose a standard preparation of
isolated pancreatic islets, because the central ß-cell-rich core of these
micro-organs undergoes rapid necrotic changes during culture times shorter
than those required for transduction and analysis of gene expression
(Ono et al., 1979
).
Furthermore, infection of isolated islets has so far resulted in a variable
cDNA targeting of those ß-cells, which are located in the centre of the
islets (Curran et al., 2002
;
Gallichan et al., 1998
;
Giannoukakis et al., 1999
;
Ju et al., 1998
;
Salmon et al., 2000
) and are
responsible for most of the acutely induced secretion
(Stefan et al., 1987
). To
bypass these limitations, still preserving the three-dimensional (3D) context
that is required for proper function of contacting islet cells
(Halban et al., 1982
;
Hauge-Evans et al., 1999
;
Hopcroft et al., 1985
), we
studied pseudo-islets formed by the spontaneous reaggregation of cells that
had been previously transduced in suspension. We found that the
glucose-induced insulin secretion of pseudo-islets was altered depending on
the type of transduced Cx and its actual level of expression.
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Materials and Methods |
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Cell cultures
RIN2A cells (Gazdar et al.,
1980) were cultured in RPMI 1640 medium containing 10% foetal calf
serum (FCS), 110 U ml1 penicillin, 110 µg
ml1 streptomycin and 2 mM L-glutamine.
Primary ß-cells, intact islets and pseudo-islets were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 11.2 mM glucose, 10% heat-inactivated FCS, 2 mM L-glutamine, 110 U ml1 penicillin and 110 µg ml1 streptomycin.
293T cells were also cultured in the DMEM medium described above, except
that the glucose concentration was 25 mM
(Naldini et al., 1996a). All
cells were kept at 37°C in a humidified incubator, gassed with air and
CO2 to maintain medium pH at 7.4.
Construction of lentiviral vectors
Three self-inactivating HIV-derived gene-transfer plasmids
(pHR'-CMV-Cx-W-sin18) were constructed by inserting the cDNA for rat
Cx32, Cx36 or Cx43 downstream of the cytomegalovirus (CMV) promoter elements
and upstream of the post-transcriptional regulatory element of woodchuck
hepatitis virus (W) (Fig. 1A).
To express Cx43, plasmid (pHR'-CMV-Cx43-W-sin18) was made by digesting
pHR'-CMV-GFP-W-sin18 (Zufferey et
al., 1999) with BamHI and XhoI, and inserting
rat Cx43 cDNA (Beyer et al.,
1987
) in place of the green fluorescent protein (GFP) cDNA. To
express Cx36, plasmid (pHR'-CMV-Cx36-W-sin18) was constructed by
digesting pHR'-CMV-Cx43-W-sin18 with SnaBI and XhoI,
and inserting the rat Cx36 cDNA
(Condorelli et al., 1998
) from
pcDNA 3.1 (Invitrogen, The Netherlands) in place of the Cx43 cDNA. To express
Cx32, plasmid (pHR'-CMV-Cx32-W-sin18) was constructed by partially
digesting pHR'-CMV-Cx43-W-sin18 with EcoRI and inserting the
rat Cx32 cDNA (Paul, 1986
) in
place of the Cx43 cDNA.
|
Production of lentiviral vectors
Vectors were prepared by co-transfecting 293T cells with the following
three plasmids at a time (Naldini et al.,
1996b): the three pHR'-CMV-Cx-W-sin18 (or the original
pHR'-CMV-GFP-W-sin18); a second-generation packaging plasmid p8.91
(Zufferey et al., 1997
); a
VSV-G envelope-protein-expression plasmid pMDG
(Naldini et al., 1996b
). After
an overnight incubation in the presence of the transfection precipitate, the
culture medium was changed. On the following day, the medium of the
transfected cells was harvested, filtered through a polyethersulfone membrane
(0.45 µm pores) and stored in 1-10 ml aliquots at 80°C. The
concentration of the vector stock [transducing infectious units (TIU)] was
determined by adding aliquots of vector on monolayers of RIN2A cells and by
assessing: (1) the percentage of GFP-positive cells by fluorescence-activated
cell sorting (FACS) using a Beckton Dickinson FACScan
(Fig. 1B) as per the guidelines
discussed by Klages et al. (Klages et al.,
2000
); (2) the immunofluorescence labelling (between neighboring
cell pairs) of different Cxs 48 hours after the infection.
Transduction of primary islet cells
Infection was carried out by adding the vectors to either established
monolayer cultures (for dye-coupling experiments) or single-cell suspensions
(for experiments involving pseudo-islets). In both cases, the infection was
performed the day following cell isolation and lasted 24 hours. Based on the
vector concentration determined on RIN2A cells (see above),
4x105 TIUs were used to infect 105 primary islet
cells with vectors encoding GFP, Cx32, Cx36 or Cx43. After several rinsing
periods, infected cells were allowed to reaggregate for 5 days. As control,
uninfected cell batches were cultured for pseudo-islet formation, in parallel
with the transduced cells. Thus, each experiment compared pseudo-islets made
of a single original batch of cells from which some aliquots were not infected
whereas others were transduced for GFP, Cx32, Cx36 or Cx43.
Immunofluorescence
For indirect immunofluorescence, pancreatic ß-cells were attached to
coverslips coated with matrix 804G (Bosco
et al., 2000) and exposed for 3 minutes to acetone at
20°C. All cells were first rinsed in cold (4°C) PBS, blocked
for 30 minutes with PBS supplemented with 2% bovine serum albumin (BSA) and
incubated for 2 hours at room temperature with one of the following
antibodies: (1) polyclonal rabbit antibodies against Cx43, diluted 1:500
(Zymed Laboratories, South San Francisco, CA); (2) polyclonal rabbit antiserum
against rat Cx32, diluted 1:400
(Dermietzel et al., 1984
); and
(3) polyclonal rabbit antiserum against rat Cx36, diluted 1:200
(Serre-Beinier et al., 2000
).
Pseudo-islets were processed in the same way except that the fixation was
performed for 1 hour with 100% ethanol at 20°C, and that the
incubation with the anti-Cx antibodies was run overnight at room
temperature.
Cells and pseudo-islets were then rinsed in PBS and incubated for 1 hour at room temperature with fluorescein-conjugated antibodies against rabbit Igs, diluted 1:500. After further rinsing, sections were stained with a 0.03% Evans' blue solution (which gives a red background staining when viewed by fluorescence using filters for fluorescein detection), covered with 0.02% paraphenylenediamine in PBS-glycerol (1:2 vol:vol) and photographed using filters for fluorescein detection with either an Axioplan microscope (Zeiss, Germany) or a confocal scanning laser microscope (LSM 510, Zeiss, Germany) equipped with a 30 mW ArKr and a 1 mW HeNe laser, and a 40x inverted objective. Optical scans were continuously collected at a scan speed of 7.2 seconds per image. Section planes were collected in 2 µm steps over a total thickness of about 40-50 µm, at both 488 nm and 543 nm excitation wavelengths. The focus, contrast and brightness settings were kept constant during image acquisition. For 3D reconstruction and analysis, images were processed for surface and alpha rendering and arranged in a movie sequence, using the LSM 510 software (Zeiss, Germany).
Western blot
For extraction of total proteins, cell cultures and control tissues were
homogenized by sonication in 0.1 M Tris-HCl, pH 7.4, supplemented with 20 mM
EDTA, 1 µg ml1 pepstatin A, 1 µg ml1
antipain, 1 mM benzamidine, 4.5 Tiu ml1 (Tiu=trypsin
inhibitor unit) aprotinin, 2 mM PMSF and 1 mM DFP, and stored at
20°C. For extraction of membrane proteins, the sonicate was
centrifuged for 10 minutes at 3000 g and 4°C, the
supernatant was collected and centrifuged for 60 minutes at 100,000
g and 4°C. Pelleted material was resuspended in PBS and
solubilized in 0.1 M Tris-HCl containing 10 mM EDTA and 20% SDS, and stored at
20°C. Protein content was measured by the DC protein assay kit
(Bio-Rad Laboratories). Aliquots of membrane or total proteins were
fractionated by electrophoresis in a 12% polyacrylamide gel and either stained
with Coomassie blue or immunoblotted, as previously described.
To this end, electrophoresed samples were transferred onto PVDF membranes
(ImmobilonTM-P, Millipore) for 2 hour in the presence of 0.01% SDS and
20% methanol, using a constant current of 400 mA. Thereafter, the membranes
were saturated for 30 minutes at room temperature in a buffer containing 10 mM
Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20 (TBS-Tween) and 5% dry milk, and
incubated overnight at 4°C with one of the following antibodies: (i) mouse
monoclonal antibodies against Cx43 (Zymed Laboratories, South San Francisco,
CA), diluted 1:500; (ii) polyclonal rabbit antiserum against rat Cx32, diluted
1:1000 (Dermietzel et al.,
1984); (iii) polyclonal rabbit antiserum against rat Cx36
(Serre-Beinier et al., 2000
),
diluted 1:400. After repeated rinsing in TBS-Tween, the immunoblots were
incubated for 60 minutes at room temperature with a goat serum against either
mouse or rabbit Igs and conjugated to horseradish peroxidase
(Bio-Rad Laboratories), diluted 1:6000. Membranes were then washed and
developed by enhanced chemiluminescence using kit ECLTM (Amersham
Pharmacia Biotech) according to manufacturer's instructions.
Dye coupling
For assessment of junctional coupling, individual cells were microinjected
by iontophoresis (Meda, 2001)
within monolayer cultures, using microelectrodes containing either 4% Lucifer
Yellow CH (Sigma Chemical, St Louis, MO) or a mixture of 5% Neurobiotin
(Vector Laboratories) and 0.4% rhodamine 3-isothiocyanate dextran 10S (Sigma)
in 150 mM LiCl. After injection of neurobiotin, cells were fixed in 4%
paraformaldehyde in phosphate buffer (0.1 M, pH 7.4) for 20 minutes, washed,
incubated in 0.25% Triton X-100 in PBS for 30 minutes, washed again and
incubated with fluorescein-conjugated streptavidin (Jackson ImmunoResearch
Laboratories), diluted 1:400 for 60 minutes.
The percentage of microinjected cells that exhibited cell-to-cell transfer of Lucifer Yellow or Neurobiotin, as well as the order of dye transfer (first order means cells contacting the microinjected cell; second and third orders means cells distant from the microinjected cell by one or two cell diameters, respectively) were determined on photographs taken either immediately after each microinjection (Lucifer Yellow) or after the streptavidin incubation (Neurobiotin).
Electron microscopy
For assessment of gap junction plaques, cells were fixed for 60 minutes in
a 2.5% glutaraldehyde solution in 0.1 M phosphate buffer, infiltrated with 30%
glycerol, frozen in Freon22 cooled in liquid nitrogen and processed for
freeze-fracture using a Balzers BAF (Balzers High Vacuum, Balzers,
Liechtenstein) (Vozzi et al.,
1995). Replicas were examined in a Philips EM 300 microscope.
Insulin secretion
To assess the secretion of insulin, batches of 50 pseudo-islets were
transferred to the chambers of a Brandel Suprafusion System (Brandel, USA) and
perfused at 37°C and at a flow rate of 500 µl min1
with a HEPES (10 mM)-buffered Krebs-Ringer buffer, pH 7.4, containing 0.1% BSA
(KRB). Each chamber was initially perfused with KRB containing 2.8 mM glucose
for 30 minutes, during which period the outflow was discarded. Pseudo-islets
were then perfused for 5 consecutive periods of 20 minutes each with KRB
containing increasing concentrations of glucose (2.8-16.8 mM) and eventually 1
mM isobutylmethylxanthine (IBMX) plus 5 µM forskolin (FSK). Insulin
secreted from the pseudo-islets into the perfusate was collected in 1 ml
aliquots. Insulin content of each aliquot was measured by radioimmunoassay
(RIA) with a charcoal separation step, using rat insulin as standard and a
guinea pig anti-rat-insulin (Linco Research) as antibody
(Meda et al., 1990).
At the end of the perfusion, the pseudo-islets were collected from each chamber and extracted in acid ethanol for determination of insulin content. Each experiments tested in parallel and simultaneously the secretory response of native pancreatic islets (not shown) and of five groups of pseudo-islets made of uninfected and infected cells (GFP-, Cx32-, Cx36- and Cx43-transduced), respectively.
Data analysis
Data were expressed as mean ± s.e.m. Differences between means were
assessed by Student's t-test and considered significant when
P<0.05.
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Results |
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Over-expression of transduced Cx increases coupling of primary islet
cells
Monolayer cultures of primary islet cells transduced using the same
protocol applied to RIN2A cells revealed that 60% of the cells featured a
green fluorescence two days after infection with a GFP-coding vector. When the
vectors contained a cDNA coding for Cx32, Cx36 or Cx43, the cognate protein
was easily detected by immunostaining in both the membrane (where
immunolabelling was consistently intense) and the cytoplasm (where
immunolabelling was of variable intensity) of most islet cells
(Fig. 2). After microinjection
of gap junction tracers, cells infected with a lentivirus encoding Cx showed
dye coupling more frequently than uninfected wild-type controls
(Fig. 3A,B). The extent of this
coupling varied depending on the tracer used and the Cx type transduced. Thus,
the intercellular exchange of Lucifer Yellow was markedly increased by
over-expression of Cx32, less by the expression of Cx43 and not by that of
Cx36 (Fig. 3A). However, the
latter transduced Cx isoform led to a sizable increase in the exchange of
neurobiotin between islet cells (Fig.
3B).
|
|
Over-expression of transduced Cx leads to the formation of gap
junctions between primary islet cells of pseudo-islets
Suspensions of single wild-type islet cells spontaneously aggregate in
vitro into 3D pseudo-islets which resemble native pancreatic islets
(Fig. 4A). This spontaneous
behaviour was not altered by transduction with lentiviral vectors coding for
GFP and the pseudo-islets observed 5 days after infection were made mostly by
cells that still expressed the transduced cDNA
(Fig. 4A,C). Islet cells
over-expressing transduced Cx32, Cx36 and Cx43 also formed pseudo-islets of
standard size (Fig. 4B,C).
Minute gap junctional plaques were detected by freeze-fracture electron
microscopy at membrane interfaces between the uninfected ß-cells of
control pseudo-islets (Fig.
5A). When the micro-organs were formed by islet cells transduced
with a lentiviral vector coding for Cx32, Cx36 or Cx43, unusual large plaques
were revealed (Fig. 5A), in
agreement with the expression of increased levels of transduced proteins
observed by immunostaining and immunoblotting
(Fig. 4B, Fig. 5B).
|
|
GFP-transduced islet cells form pseudo-islets that secrete
normally
Total insulin content of GFP-transduced pseudo-islets (1832±403 ng
per chamber, n=4) was similar to that of uninfected pseudo-islets
(1683±371 ng per chamber, n=5)
(Fig. 6B). Uninfected
pseudo-islets increased (P<0.05) insulin secretion twofold over
the basal level observed in the presence of 5.6 mM glucose, when the
concentration of the sugar was raised to 16.8 mM glucose
(Fig. 6A,C). Addition to the
sugar of 1 mM IBMX and 5 µM FSK further potentiated the insulin output,
which was then increased eightfold (P<0.05) over that observed
under basal conditions (Fig.
6A,C). Similar results were obtained with pseudo-islets made of
GFP-transduced cells (Fig.
6A,C). Thus, when compared with pseudo-islets made of uninfected
cells, pseudo-islets made of GFP-transduced cells did not show any alteration
in insulin secretion, whether the hormone release was assessed after
stimulation by glucose or by drugs that potentiate the sugar effect by raising
the cytoplasmic concentration of cAMP (Fig.
6A).
|
Pseudo-islets made of Cx-transduced cells show normal insulin content
but altered release of the hormone
Total insulin content of Cx-transduced pseudoislets (1480±263 ng per
chamber, n=12; data from all Cx-transduced cells pooled) was similar
to that of control pseudo-islets (1749±411 ng per chamber,
n=9; data from GFP-transduced and uninfected cells pooled)
(Fig. 7A). Pseudo-islets made
of Cx43-transduced cells also featured a control secretory response (i.e.
increasing secretion in the presence of 16.8 mM glucose)
(Fig. 7B). By contrast,
pseudo-islets made of cells transduced with either Cx32 or Cx36 failed to
increase significantly their insulin release over the basal level when
stimulated by 16.8 mM glucose (Fig.
7B). Under this condition, their insulin output was 48.5±5%
(Cx32, n=3, P<0.005) and 51±5% (Cx36,
n=3, P<0.005) the control values (data of GFP-transduced
and uninfected pseudo-islets pooled), respectively
(Fig. 7C). All Cx-transduced
pseudo-islets significantly increased their insulin release after stimulation
by glucose plus IBMX and FSK (Fig.
7B). However, this increase was smaller than that of controls,
representing 58±3%, 67±17% and 71±16% of the normalised
control value, in pseudo-islets transduced for Cx32 (n=3;
P<0.05), Cx36 (n=3) and Cx43 (n=3)
(Fig. 7C).
|
![]() |
Discussion |
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To bypass these difficulties, we have designed a novel generation of
lentiviral vectors to transduce distinct Cx cDNAs in cells of the
insulin-producing RIN2A line (Gazdar et
al., 1980) which, in the wild-type configuration, do not
transcribe Cx genes (Vozzi et al.,
1995
). Here, we document that these vectors efficiently
upregulated the expression of Cx proteins in RIN2A cells and that the
transduced connexins had a normal molecular weight and epitope structure. We
further show that the transduced proteins were appropriately targeted to the
membrane and became normally concentrated at sites of cell-to-cell contact,
ultimately resulting in the development of bona fide gap junction plaques.
In a second step, we have shown that the same lentiviral vectors also
efficiently transduced Cx cDNAs in primary islet cells, allowing
over-expression of the cognate proteins. These proteins formed channels that
significantly increased the exchange of different gap-junction-permeant
tracers between ß-cells. The degree of coupling, however, varied
according to the gap junction tracer used and depended on both the type and
level of the Cx expressed. Thus, exchange of Lucifer Yellow [a negatively
charged molecule whose hydrated diameter approximates that of the Cx pore
(Harris and Bevans, 2001)] was
observed to spread up to three orders of islet cells after transduction of
Cx32 and Cx43, whereas it was consistently restricted to the first order of
cells in control cultures and after over-expression of Cx36. Moreover, the
exchange of neurobiotin, a positively charged molecule that is smaller than
Lucifer Yellow (Harris and Bevans,
2001
) and spreads as little as this tracer between control islet
cells, increased up to three orders of cells after transduction of Cx36. Thus,
over-expression of distinct Cxs provided increased islet cell coupling
whichever connexin isoform was over-expressed, and led to a change in the
specificity of the molecular transfer when Cx32 or Cx43 were involved. Hence,
the lentiviral vectors we have generated open new perspectives for
investigating Cx-dependent effects in primary cells that are not easily
amenable to the experimental expression of foreign genes. Indeed, these
vectors govern the stable transduction of normal cells, irrespective of their
type and cycling status (Naldini et al.,
1996b
) and might be designed for both over-expression and
downregulation of Cx proteins.
We have used these vectors to study the effect of various Cx changes on the
main physiological function of ß-cells, which is to synthesize, store and
release insulin in a regulated way. Because these processes are multicellular
functions that take place in a 3D context, we have first tested the transduced
cells for their ability to reaggregate spontaneously into pseudo-islets, which
feature morphological and functional characteristics reminiscent of those of
native pancreatic islets (Halban et al.,
1987; Hopcroft et al.,
1985
). We have found that transduction of a Cx-unrelated protein
(GFP) did not impair the spontaneous ability of islet cells to aggregate into
glucose-sensitive pseudo-islets. We have further observed that the functioning
of these micro-organs which, under control conditions, expressed low levels of
the islet native Cx36, was not affected by over-expression of Cx43, a protein
whose presence in pancreatic islets is disputed, at least in control rodents
(Charollais et al., 1999
;
Collares-Buzato et al., 2001
;
Meda et al., 1993
;
Theis et al., 2001
). By
contrast, we have observed that over-expression of either Cx32 or Cx36 reduced
the response of pseudo-islets to glucose. Thus, in spite of a control content
of insulin, the latter pseudo-islets were unable to enhance the release of the
hormone in response to an increase in the glucose concentration of the
perfusate. All types of Cx-over-expressing pseudo-islets significantly
increased their insulin secretion over basal values in response to stimuli
raising the intracellular concentration of cAMP
(Gillis and Misler, 1993
),
even though somewhat less than controls. These findings show that the loss of
glucose responsiveness observed in the same pseudo-islets cannot be accounted
for by a nonspecific inhibition of the secretory machinery of
ß-cells.
Because these secretory alterations were paralleled by changes in the
intercellular transfer of molecules that had a size and charge comparable to
those of many endogenous metabolites
(Bevans et al., 1998;
Harris and Bevans, 2001
;
Veenstra et al., 1995
), it is
plausible that they resulted from the abnormal ß-cell-to-ß-cell
exchange of some gap-junction-permeant signal. Ca2+ is a probable
candidate, because this cation plays an important role in insulin secretion,
diffuses through junctional channels and features abnormal stimuli-induced
oscillations after Cx alterations
(Charollais et al.,2000
;
Calabrese et al., 2003
).
Accordingly, the secretory defect we observed in this study is reminiscent of
that observed in transgenic mice whose ß-cells over-expressed Cx32. Under
these conditions, the induced Cx32 channels improved the electrical
synchronisation of ß-cells and the glucose-induced increase in the
intracellular concentration of Ca2+
(Charollais et al., 2000
). In
spite of these changes, that would be expected to promote insulin release
(Bergsten, 2000
;
Kanno et al., 2002
;
Satin, 2000
), the secretion of
the hormone was not stimulated by glucose, indicating that the signal(s) whose
exchange had been increased between ß-cells negatively affected secretion
(Charollais et al., 2000
).
Even though the nature of this signal remains to be elucidated, our data
provide evidence that it does not transfer similarly from cell-to-cell
whatever the Cx involved. Specifically, the data show that, in this respect,
Cx32 channels cannot substitute the native Cx36 channel. In this perspective,
it remains to be understood why over-expression of Cx36 also resulted in
impaired glucose-induced insulin secretion. Because this over-expression
increased the cell-to-cell exchange of some (e.g. neurobiotin) but not all
gap-junction-permeant molecules (e.g. Lucifer Yellow), which is consistently
restricted to small territories of ß-cells within native islets
(Charollais et al., 2000;
Michaels and Sheridan, 1981
),
it is conceivable that extended coupling caused an excessive dilution of the
Cx-dependent signals that positively control secretion. However, our data show
that, even in this case, the specific characteristics of individual Cxs
matter, in as much as no deleterious secretory effect was observed after
transduction of Cx43, in spite of the fact that this protein increased
coupling up to the levels elicited by the Cx32 and Cx36 transduction. The
findings are consistent with the different molecular permeability of channels
made by distinct Cx isoforms (Bevans et
al., 1998
; Goldberg et al.,
2002
). They imply that the signals diffusing through Cx43 channels
are required for the cAMP-dependent potentiation of insulin release but not
for the glucose stimulation of this event. Conversely, signals diffusing
through either Cx32 or Cx36 channels contribute to modulate the two forms of
insulin secretion. Additional experiments, in which different levels of
specific Cx isoforms are compared in the absence of other connexins, will now
be required unambiguously to establish the relative contributions to this fine
tuning of the amount of each Cx isoform, on the one hand, and of its specific
conductance and permeability characteristics, on the other. Addressing this
important question might help us to identify the still-elusive
gap-junction-permeant signals that influence insulin secretion
(Meda and Spray, 2000
;
Serre-Beinier et al., 2003). As a first approach to this central issue, we
have recently shown an essential role of Cx36 channels in synchronizing
stimulus-induced Ca2+ oscillations between the insulin-producing
cells of the MIN6 line (Calabrese et al.,
2003
).
In summary, our study first shows that lentiviral vectors are efficient
tools to modulate the levels of Cxs and coupling in primary, non-dividing
cells. With the availability of these tools, several questions about the
(patho)physiological function(s) of gap junction proteins
(Meda and Spray, 2000) can now
be taken to direct experimental testing in primary tissues. In this
perspective, we document that appropriate levels of specific Cx isoforms
selectively influence distinct aspects of insulin secretion of primary
pancreatic ß-cells. Lentiviral vectors have also been shown to have some
potential for gene therapy (Gallichan et
al., 1998
; Kordower et al.,
2000
). Therefore, their usefulness in a future correction of
hereditary, Cx-linked diseases (Kelsell et
al., 2001a
; Kelsell et al.,
2001b
) should be envisaged.
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Acknowledgments |
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References |
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