1 Department of Human Morphology, Faculty of Medicine
2 Department of Biology, Faculty of Arts and Sciences, American University of
Beirut, PO Box 11-0236, Beirut, Lebanon
* Authors for correspondence (e-mail: me00{at}aub.edu.lb; rtalhouk{at}aub.edu.lb)
Accepted 7 May 2003
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Summary |
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Key words: Connexins, Differentiation, ECM, GJIC, Mammary
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Introduction |
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Besides humoral mediators (Hynes et
al., 1997; Hennighausen et
al., 1997
), the extracellular matrix (ECM) has been regarded as
the dominant regulator of mammary differentiation
(Weaver et al., 1997
;
Boudreau and Bissell, 1998
;
Schmeichel et al., 1998
;
Smalley et al., 1999
;
Klinowska and Streuli, 2000
;
Hansen and Bissell, 2000
). The
few studies that addressed the role of cell-cell interaction in mammary
differentiation (Streuli et al.,
1991
; Desprez et al.,
1993
; Alford and
Taylor-Papadimitriou, 1996
;
Hansen and Bissell, 2000
)
undervalued its role as compared with the effect exerted by the ECM.
Studies have suggested that gap junctions play a critical role in the
coordinated changes through development, differentiation, maintenance and
involution of the mammary gland (Monaghan
et al., 1994; Monaghan et al.,
1996
; Pozzi et al.,
1995
; Yamanaka et al.,
1997
; Locke et al.,
2000
; Yamanaka et al.,
2001
). However, no studies have established a clear correlation
between functional GJIC and mammary epithelial differentiation, either in vivo
(Perez-Armendariz et al.,
1995
; Pozzi et al.,
1995
; Monaghan and Moss,
1996
; Locke et al.,
2000
) or in vitro (Lee et al.,
1991
; Lee et al.,
1992
; Tomasetto et al.,
1993
; Hirschi et al.,
1996
; Sia et al.,
1999
).
The CID-9 mouse mammary cell culture system, responsive to both lactogenic
hormones and substrata, consists of a heterogeneous cell strain of epithelial,
myoepithelial and fibroblastic cells and is a widely accepted model that
mimics in vivo differentiation of mammary cells
(Schmidhauser et al., 1992;
Talhouk et al., 2001
). To
determine the role of GJIC in modulating the differentiation phenotype of
mammary cells, gap junction proteins of mammary CID-9 cells were characterized
and their regulation by ECM assessed. The cause-and-effect relationship
between GJIC and mammary epithelial differentiation was also investigated. We
demonstrate that mammary CID-9 cells express Cx26, Cx32 and Cx43 proteins,
which are modulated by ECM, and that proper cell-ECM interaction favours GJIC.
Our studies suggest CID-9 cells are capable of differentiating and expressing
ß-casein in the absence of an exogenous basement membrane in a
ß1-integrin-independent pathway, provided the cells are
coupled via functional gap junctions.
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Materials and Methods |
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Cell culture
A low passage number (17 to 21) of the CID-9 mouse mammary cell strain was
used throughout. Cells were grown in 'growth medium' consisting of Dulbecco's
Modified Eagle's Medium Nutrient Mixture F12 Ham (DMEM/F12) with 5% FBS,
insulin (5 µg/ml) and gentamycin (50 µg/ml) in a humidified incubator
(95% air 5% CO2) at 37°C. Cells were propagated by
trypsinization and plated either on tissue culture plastic petri dishes or on
petri dishes coated with different substrata.
CID-9 cells were seeded at 3.0x106 or at
5.0x106 cells/75cm2 dish on culture dishes or
dishes coated with the reconstituted basement membrane, growth-factor-reduced
Matrigel, respectively. Alternatively, diluted Matrigel (1.5% vol/vol) in HBSS
was dripped onto cells 24 hours after plating
(Streuli et al., 1995a). Cells
cultured on EHS-matrix were directly plated in differentiation or
non-differentiation media consisting of DMEM/F12 containing insulin (5
µg/ml), hydrocortisone (1 µg/ml) and either supplemented with or lacking
ovine prolactin (3 µg/ml), respectively. Cells cultured on plastic or
dripped with EHS-matrix were first plated in growth medium for initial cell
attachment and spreading. Twenty-four hours after plating, cells were washed
three times with HBSS, and the growth medium was replaced with either
differentiation or non-differentiation media. Media were changed on a daily
basis.
PolyHEMA, a non-adhesive substratum, was prepared using a solution of 6 mg/ml in 95% ethanol and was added to culture plates at 5.0x10-2 ml/cm2 and allowed to evaporate to dryness at 37°C. The plates were then washed twice with HBSS and CID-9 cells were plated at a concentration of 5.0x105 cells/ml and in differentiating media. Since the cells were grown in suspension, cAMP diluted in differentiating media was added to the existing media on days 1, 3 and 5 of culture. On day 6 of culture, the cells were harvested for analysis.
RNA extraction and northern blot analysis
Total RNA was extracted from cells at day 6 after plating as described
elsewhere (Chomczynski and Sacchi,
1987). For northern analysis, 5 µg of total RNA were
electrophoresis through 1% agarose/formaldehyde gel, blotted overnight onto
Amersham Hybond-N membrane in 10x SSC and UV crosslinked for subsequent
hybridization. ß-Casein c-DNA inserts were 32PdCTP-labelled
using Rediprime kit and hybridization was performed overnight at 42°C in a
shaker-incubator. The blots were then washed at high stringency (0.1% SSC,
65°C) and signals were detected by fluorography.
Western blot analysis
Proteins were extracted by scraping the cells into lysis buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate). The
scraped cells were then sheared by passing them several times through a
21-gauge needle. Protease inhibitors were added at a concentration of 40 µl
per 1 ml of lysis buffer, and the cell extracts were centrifuged. The protein
content of the supernatants was determined by Bio-Rad assay and equal amounts
of protein were resolved by gel electrophoresis. To detect milk proteins, the
membranes were blocked overnight in a wash buffer (100 mM Tris-HCl buffer, pH
7.5, 150 mM NaCl. 0.3% Tween 20) with 2% fatty acid-free BSA. The membranes
were then incubated for 1 hour in polyclonal rabbit anti-mouse milk antiserum
at room temperature and washed three times, for 20 minutes each, to remove
unbound antiserum.
For Cx26, Cx32 and Cx43 proteins, membranes were blocked for 1 hour in a wash buffer [Dulbecco's phosphate buffered saline (PBS), 0.1% Tween 20] with 3% skim milk. They were then incubated for two hours in a humid chamber in the corresponding polyclonal rabbit anti-Cx antibody at a concentration of 0.5 µg per ml of blocking buffer. Bound antibody was detected by enhanced ECL for casein, Cx32 and Cx43 immunoblots. Addition of HRP-conjugated anti-rabbit IgG followed by tetramethyl benzidine (TMB) was used for detection of Cx26 immunoblots.
Immunohistochemistry
Cultured CID-9 cells were washed three times with warm HBSS and fixed in
ice-cold (-20°C) 70% ethanol overnight. Fixed cells were first rinsed
twice with PBS and then incubated for 1 hour at room temperature with 3%
normal goat serum. After blocking, cells were labelled for 2 hours at room
temperature with rabbit anti-connexin 26, 32 and 43. This was followed by
labelling with a FITC-conjugated secondary goat anti-rabbit IgG (H+L) that was
incubated for 1 hour with the fixed cells. Concentrations of the primary and
secondary antibodies were used as recommended by the supplier. Nuclei were
then counter-stained by incubation for 3 minutes with propidium iodide at 5
µg/ml. Washing with PBS was performed twice between incubations. Finally,
cells were mounted on slides and staining was preserved by addition of
antifade to the stained cells, which were kept at 4°C. Cells were then
observed under fluorescence microscopy (LSM 410, Zeiss, Germany).
Lucifer yellow (LY) dye microinjection and scrape-loading assays
For microinjection assay, CID-9 cells were cultured on MatTek glass bottom
tissue culture plates. Cells were microinjected with 5% LY CH in 150 mM LiCl.
The solution of LY was injected by pressure injection into cells through
microelectrodes. The spread of the dye fluorescence to neighbouring cells was
recorded photographically using fluorescence microscopy.
For scrape-loading assay, CID-9 cells were cultured on plastic or on
EHS-drip, into 4-chamber polystyrene vessel tissue culture-treated glass
slides. After 24 hours, medium was supplemented with 10 µM 18 GA, or
50 µM 8-Br-cAMP.
The scrape-loading method was performed as described elsewhere
(El-Fouly et al., 1987). Cells
plated on 4-chamber vessel slides were washed three times with warm HBSS
before addition of LY at 0.1% dilution in PBS. Using a scalpel, cuts were made
throughout the monolayer, followed by incubation for 10 minutes at 37°C.
The cells were then washed with warm HBSS and fixed with 4% formaldehyde.
Slides were preserved by mounting in antifade and stored at 4°C.
Observation of dye spread was recorded photographically as described
earlier.
18 GA and 8-Br-cAMP treatment of CID-9 cells
CID-9 cells were seeded on EHS-matrix and treated with 10 µM 18
GA on day 1 of culture. Medium supplemented with 18
GA was changed on a
daily basis up to day 10 in culture. Alternatively, 18
GA was
supplemented to the medium for the first 5 days in vitro and later removed
from the medium for days 6-10. Trypan Blue staining was used to determine cell
viability as affected by 18
GA for the duration of the treatment.
Samples were counted in triplicate wells. Proteins for western blot analysis
(normalized to equal cell counts) were extracted on day 5 and day 10 of
culture. Control cells were not treated with 18
GA.
CID-9 cells treated with 8-Br-cAMP were seeded in 100 mm petri dishes on tissue culture plastic. Twenty-four hours later, growth medium was replaced with differentiation medium and 8-Br-cAMP was added at a concentration of 50 µM. Medium supplemented with 8-Br-cAMP was changed on a daily basis up to day 5 of culture, when proteins for western blot analysis were extracted. Control cells were not treated with 8-Br-cAMP.
Treatment of CID-9 cells with integrin function-blocking
antibody
CID-9 cells were seeded in 6-well plates on plastic substratum or EHS-drip.
Twenty-four hours after plating, cells were washed three times with HBSS and
growth medium was replaced with differentiation medium containing 100 µg/ml
of the function-blocking ß1 integrin antibody. The antibody
was supplemented daily with the media and the cells were harvested at day 4
after plating.
Quantitative analysis of ß-casein, connexin expression and
functional GJIC
ß-Casein, connexin expression and functional GJIC using LY scrape-load
assays were quantified from different experiments using NIH Image 1.62
software. Quantification of ß-casein and connexin expression was
normalized with respect to ß-actin. For LY scrape-load assays,
quantification was based on measuring the integrated fluorescence intensity,
at the scrape site, over an equivalent area in both control and experimental
conditions. The degree of significance of variations between control and
experimental values was assessed by ANOVA uni-variant test using the Graph Pad
Prism software version 3.00. Where applicable, the quantification was from
three different experiments.
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Results |
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To determine the effect of substratum on expression of connexin proteins by CID-9 cells and their subcellular distribution, western blot analysis and immunolocalization studies were performed. Whereas no significant change was noted in Cx32 expression, slight but significant (P<0.05) upregulation of Cx26 expression in CID-9 cells was noted on EHS-drip as compared with cells on plastic. All Cx43 isoforms (NP: non-phosphorylated inactive form; P1 and P2: phosphorylated, active forms) were expressed on plastic, EHS-drip and on EHS-matrix on day 6 of culture. However, on EHS-matrix, the P2 form of Cx43 protein became predominant and was significantly (P<0.001) elevated compared with the levels detected on plastic or EHS-drip (Fig. 2A).
|
To assess the relationship, if any, between connexin intercellular distribution and cell differentiation, immunohistochemistry was employed to grade for connexin cytosolic or plasma membrane distribution on either plastic or EHS-drip after 6 days of culture. Cx26, Cx32 and Cx43 distribution was mainly cytosolic when cells were grown on plastic (Fig. 2Ba, Bc, Be). In certain limited areas of the culture, Cx26, Cx32 or Cx43 localized to the plasma membrane of cells on plastic (Fig. 2Ba, Bc, Be; inserts). Connexin distribution became predominantly membranous when cells were cultured on EHS-drip (Fig. 2Bb, Bd, Bf). Note that certain cells, as evident by immunostaining, did not express Cx26 and Cx43 under any of the culture conditions studied. Whether these cells are fibroblastic, myoepithelia or epithelial was not addressed in our studies.
The change in connexin distribution from intercellular on plastic to plasma membrane for cells grown on EHS-drip compared with those on plastic strongly suggests that communication via gap junctions is enhanced by cell-ECM interaction. To test for gap junction functionality, cells were injected with LY or scraped, and transfer of fluorescent dye into neighbouring cells was observed. In confluent cultures of CID-9 cells plated on plastic, dye transfer was limited to a few cells neighbouring the dye-injected cell (Fig. 3A); similarly, dye transfer was restricted to 1-2 layers deep beyond the scrape site (Fig. 3B). However, on EHS-drip, LY was transferred to several cells immediately neighbouring the microinjected cell (Fig. 3C) and beyond 4-5 cell-layers deep away from the scrape site (Fig. 3D). Quantitative analysis, as described in the Materials and Methods, of the integrated fluorescence intensity at the scrape site from three different experiments showed an increase in fluorescence from 36±10 for cells on plastic to 132±36 for cells on EHS-drip. This demonstrated that cells on EHS-drip communicate via gap junctions more readily than cells on plastic.
|
Modulation of GJIC affects mammary epithelial differentiation
To determine if gap junctional communication is necessary for ß-casein
expression, two approaches were undertaken. In the first, 18 GA was
utilized to inhibit gap junctional communication of cells cultured on
EHS-matrix. In the other, cAMP was utilized to enhance gap junctional
communication by cells on plastic. In contrast to control cells, those cells
treated with 10 µM 18
GA and cultured on EHS-drip showed disrupted
GJIC, and downregulated their Cx43 and ß-casein expression without
drastically affecting cell viability as determined by Trypan Blue staining
(Fig. 4A). Disrupting GJIC also
affected the clustering behaviour of CID-9 cells grown on EHS-matrix, yielding
smaller size clusters that lacked well-defined membrane-like boundaries
(Fig. 4Bb compared with
Fig. 4Ba). ß-Casein
protein expression on day 6 of 18
GA-treated culture was dramatically
decreased compared with the control (upto 95%) non-treated cells. The effect
of the gap junction inhibitor 18
GA was reversible. Adding media devoid
of 18
GA beyond day 6 showed that partial recovery of clustering
morphology was noted (Fig.
4Bc). ß-Casein expression was also partially (approximately
50%) recovered 4 days after the depletion of 18
GA from the medium
(Fig. 4C).
|
cAMP-treated CID-9 cells plated on plastic showed a threefold enhanced LY transfer beyond 2-3 cell-layers deep away from the scrape site. cAMP also induced aggregation of CID-9 cells, resulting in enhanced clustering (Fig. 5A). Northern and western blot analyses showed that cAMP-treated CID-9 cells on plastic expressed ß-casein mRNA and proteins (Fig. 5B,C) as well as higher levels of Cx26 and Cx43 proteins on day 6 of culture compared with the non-treated control cells grown in parallel (Fig. 5C).
|
Although cAMP enhanced Cx43 and Cx26 expression and upregulated gap
junctional communication as well as ß-casein expression, it was not clear
whether casein expression was owing to enhanced gap junctional communication
or other cAMP-mediated effects. To assess this, cells were treated with both
cAMP and 18 GA. This treatment induced downregulation of ß-casein
(Fig. 6A).
|
To find out whether the cAMP effect on CID-9 cells may also require cell-ECM interaction, cAMP-treated cells were incubated with function-blocking ß1-integrin antibody. Whereas, in cAMP-treated cultures, ß-casein expression was partially downregulated by ß1-integrin function-blocking antibody, this treatment markedly (P<0.01) decreased ß-casein production in cells cultured on EHS-drip (Fig. 6B). This suggested that, in the presence of cAMP, treated cells, with enhanced GJIC, can express ß-casein in a ß1-integrin-independent manner.
cAMP also induced differentiation of CID-9 cells in suspension independently of a cell-ECM interaction. CID-9 cells were plated on the non-adhesive polyHEMA substratum and treated with cAMP. Cells on PolyHEMA grew in small clusters not exceeding 5-6 cells in each cluster and did not express ß-casein. By contrast, cells on PolyHEMA treated with cAMP aggregated into larger clusters, and upregulated their Cx43 expression. These cells also expressed ß-casein (Fig. 7).
|
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Discussion |
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Evidence from the literature has shown that the main role of cell-cell
interactions is to enhance mammary cell differentiation, but in a
matrix-dependent manner (Emerman et al., 1977;
Lee et al., 1985;
Streuli and Bissell, 1990
;
Petersen et al., 1992
;
Desprez et al., 1993
;
Talhouk et al., 1993
;
Slade et al., 1999
). A study
by Streuli et al. emphasized the importance of cell-cell interaction in
mammary cell differentiation when they demonstrated that casein production was
synergistically elevated upon cell-cell interaction
(Streuli et al., 1991
). A
consequence of cell-cell interaction could be intercellular communication
through gap junctions. Although the importance of gap junctions is well
established in the differentiation of many cell types
(Pitts et al., 1988
;
Paul et al., 1995
;
Bruzzone et al., 1996
;
Kumar and Gilula, 1996
;
Wiszniewski et al., 2000
;
Alford and Rannels, 2001
;
Gramsch et al., 2001
;
Romanello et al., 2001
;
Schiller et al., 2001
), few
studies have characterized gap junctions in the mammary gland. The major
connexins described in the mammary gland are Cx26, Cx32 and Cx43
(Lee et al., 1991
;
Lee et al., 1992
;
Tomasetto et al., 1993
;
Perez-Armendariz et al., 1995
;
Pozzi et al., 1995
;
Monaghan and Moss, 1996
;
Yamanaka et al., 1997
;
Sia et al., 1999
;
Yamanaka et al., 2001
), but no
studies have established a clear correlation between connexin expression,
functional GJIC and mammary epithelial differentiation. In this study, we
provided evidence for the importance of intercellular interaction for
targeting and functionality of connexins in mammary cells, and the
differentiated phenotype resulting thereof.
The use of northern and western blot analysis coupled with
immunolocalization studies established that CID-9 cells expressed and
modulated all major mammary gland connexins and provided better insight to the
locale and, hence, the role of connexin in the differentiation of mammary
epithelial cells. The regulation of connexin expression by the ECM has been
reported by Guo et al. (Guo et al.,
2001), whereby lung alveolar type II epithelial cells modulated
the expression and distribution of Cx43 and Cx26 depending on the substrata
the cells were plated on. The downregulation of Cx43 mRNA but not Cx26 mRNA in
differentiated CID-9 cells cultured on EHS-matrix was reminiscent of earlier
studies (Rosenberg et al.,
1996
), whereby Cx26 and Cx43 transcripts declined whereas those of
Cx32 increased as hepatic cells differentiated. In this study, the P2
phosphorylated form of Cx43 was upregulated in cells on EHS-matrix, suggesting
that Cx43 regulation during cellular differentiation occurred at the
post-translational level through phosphorylation. This correlated with in vivo
studies (Yamanaka et al.,
1997
), and was supported by data from our laboratory demonstrating
that, during lactation, Cx43 mRNA in the mammary gland is downregulated
whereas the P2 form of Cx43 protein is upregulated (R.S.T. et al.,
unpublished).
On day 6 of culture on EHS-drip, Cx26, Cx32 and Cx43 localized mostly to
the plasma membrane. By contrast, connexins localized mostly to the cytoplasm
in cells cultured on plastic. Studies performed on human luminal and basal
mammary cells in culture revealed mostly cytoplasmic, rarely membranous, Cx26
staining associated with luminal epithelial cells
(Monaghan et al., 1996). Cx43
localized mostly to membranes of cells with large nuclei of the CID-9 cell
population that stain positive for smooth muscle actin. These represent the
myoepithelial-like subpopulation of CID-9 cells
(Desprez et al., 1993
). Other
studies (Monaghan et al.,
1996
; Yamanaka et al.,
1997
; and Locke,
1998
) reported Cx43 localization to myoepithelial cells. Cx26 and
Cx32 immunostaining had no distinct cell-type distribution. These data
supported by gap junction functionality assays, suggested that at least Cx26,
Cx32 and Cx43 redistributed and assembled, by day 6 in culture, into
communicating junctions due to matrix-dependent post-translational
modifications. Similar data were reported for redistribution of intracellular
stores of Cx43 in a quiescent MAC-T bovine mammary epithelial cell line
(Sia et al., 1999
) and in
alveolar epithelial cells as demonstrated by Guo et al.
(Guo et al., 2001
). It is
difficult to rule out the potential involvement of other connexins that we did
not assay for.
Thus, differentiation is coupled to enhanced connexin membrane localization
and GJIC. However, whether gap junctions actually mediate the differentiation
process and/or whether the differentiation process depends solely on
ECM-transduced signals is not clear. The differentiation re-acquired by CID-9
cells on EHS-matrix upon withdrawal of 18 GA suggested that cell-ECM
interaction was not sufficient to maintain an optimal differentiated
phenotype, whereas proper cell-cell and cell-ECM interaction were crucial for
optimal differentiation. Many studies reported that 18
GA blocked GJIC
in different cell types (Martin et al.,
1991
; Taylor et al.,
1998
; Venance et al.,
1998
) and others have shown that 18
GA rapidly and
reversibly blocked GJIC and that extended exposure to 18
GA inhibited
both Cx43 mRNA and protein expression in a time- and dose-dependant manner
(Guo et al., 1999
). In the
present study, we showed that 18
GA downregulated Cx43 expression. The
mechanism of action of 18
GA on connexin expression requires further
elucidation.
Regulation of gap junctions by cAMP has been demonstrated at the
transcriptional, translational and post-translational levels. The latter was
mediated by phosphorylation of connexin proteins, by redistribution of gap
junction plaques to the cell membrane, or even by increase in the rate of
trafficking of individual connexins to the membrane
(Atkinson et al., 1995;
Paulson et al., 2000
;
Lampe et al., 2000
;
Lampe and Lau, 2000
). In this
study, we showed that cAMP-enhanced GJIC was coupled to increased Cx43
expression and its phosphorylation as well as increased Cx26 expression by
cells on plastic. This was in agreement with other studies
(Furger et al., 1996
) where
cAMP treatment of human granulosa cells enhanced GJIC by increasing the levels
of Cx43 phosphorylation. Most importantly, enhanced GJIC due to cAMP was
implicated in CID-9 differentiation even in the apparent absence of an
exogenous basement membrane. In addition to the increased ß-casein
production, cAMP led to a decrease in the production of laminin and collagen
IV (Sfeir et al., 2001
).
Pervious studies (Streuli and Bissell,
1990
) showed that expression of laminin and type IV collagen were
lowest in culture conditions that favoured ß-casein expression. However,
this correlated with deposition of a continuous basement membrane. The
increased ß-casein expression in our studies could have thus occurred in
a gap-junction-independent pathway and as a result of the endogenously
deposited basement membrane as suggested by previously
(Petersen et al., 1992
).
However, the loss of differentiation as well as Cx43 expression by
cAMP-treated cells together with a gap junction inhibitor, 18
GA,
suggested that the effect of cAMP on ß-casein expression and mammary
differentiation was achieved through a gap junctional communication manner.
The concentration of 18
GA and the duration of its use were not
cytotoxic to cells (Davidson et al.,
1986
), and showed no drastic effect on cell viability; hence, the
downregulation of ß-casein expression in CID-9 cells was not due to
nonspecific effects. Moreover, we suggest that heterocellular gap junction
communication is a critical element in mediating mammary epithelial
differentiation, since 18
GA inhibited the ß-casein (R.S.T. et
al., unpublished) that is typically expressed in co-cultures of SCp2 and SCg6
cells on plastic in the absence of exogenously provided basement membrane
(Desprez et al., 1993
).
We confirmed that ß-casein expression by CID-9 cells on EHS-drip was
disrupted, in a dose-dependent manner (data not shown), by function-blocking
anti-ß1-integrin antibody at concentrations previously shown
to markedly affect ß-casein expression
(Streuli et al., 1995b;
Muschler et al., 1999
).
cAMP-treated cells cultured on plastic and incubated with function-blocking
anti-ß1-integrin antibody showed only a marginal decrease in
ß-casein production, suggesting that enhanced GJIC can induce partial
differentiation in the absence of cell-ECM interaction. Studies
(Roskelley et al., 1994
) have
indicated the presence of two classes of signals that are generated by the
ECM. The physical signals that involve cell rounding and clustering and are
mimicked by plating the cells on PolyHEMA, and the
ß1-integrin-mediated biochemical signals. CID-9 cells
expressed ß-casein on PolyHEMA only when supplemented with cAMP,
suggesting that enhanced GJIC lead to ß-casein expression on a
non-adhesive substratum by substituting for the
ß1-integrin-mediated biochemical pathway.
In conclusion, these studies demonstrated that cell-cell communication via gap junctions is essential for growth and differentiation of mammary epithelial cells in vitro. Moreover, for the first time, it is reported that cell-cell communication via gap junctions was able to initiate a partial differentiation process on a non-adhesive substratum and in the absence of an exogenous basement membrane. Finally, cell-ECM interaction alone was not sufficient to induce an optimal differentiation phenotype.
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Acknowledgments |
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