Cell Biology International 30 (2006) 472e479www.elsevier.com/locate/cellbi

Differential expression of connexin 43 in mouse mammary cells

Teresa Lambe 1, Darren Finlay 2, Madeline Murphy, Finian Martin*

UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

Received 18 January 2005; revised 7 April 2005; accepted 11 July 2005

Abstract

In this study we have employed suppressive subtractive hybridization (SSH) analysis to investigate differential gene expression in primarymouse mammary epithelial cells (PMMEC) cultured under mildly apoptotic/quiescent and differentiating conditions. Among a small group ofgenes whose expression was differentially regulated was connexin 43. In vitro, connexin 43 mRNA and protein were detectable in PMMECcultured under proliferative or mildly apoptotic conditions. The level of connexin 43 mRNA expression in vivo was also investigated. High levelsof expression were found to be associated with the periods of greatest glandular plasticity (pubertal expansion of the mammary tree, early preg-nancy and during early involution). Thus, terminally differentiated cells in vivo and in vitro did not express connexin 43 mRNA suggesting thatconnexin 43 expression, and perhaps facilitated gap junction communication, is associated with undifferentiated progenitor cell populations.� 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Gene expression; Mouse mammary gland; Connexin 43; Primary mammary cell culture

1. Introduction

The microenvironment of a cell determines its fate througha number of factors including cellecell and celleextracellularmatrix (ECM) interactions, both of which are required to main-tain cellular integrity. Dissecting the individual role of each path-way is complex in a complete organ such as the mouse mammarygland. However, culturing primary cells offers a simpler systemin which to examine the relative contribution of each pathwayand can offer insights into the complex in vivo situation.

The isolation, and short term propagation, of PMMECcultures is an established technique which has contributedtowards a functional understanding of the intact organ(Marshman et al., 2003; Srebrow et al., 1998). In vitro, the ab-sence of an ECM overlay results in undifferentiated PMMECwhich are prone to apoptose but the presence of insulin and

* Corresponding author. Tel.: þ353 1 716 6734; fax: þ353 1 269 2749.

E-mail address: finian.martin@ucd.ie (F. Martin).1 Present address: The Henry Wellcome Building of Molecular Physiology,

Roosevelt drive, Oxford OX3 7BN, UK.2 Present address: The Burnham Institute, 10901 North Torrey Pines Rd., La

Jolla, CA 92037, USA.

1065-6995/$ - see front matter � 2006 International Federation for Cell Biology.

doi:10.1016/j.cellbi.2006.02.008

a laminin-rich ECM overlay promotes cell survival (Boudreauet al., 1995; Pullan et al., 1996; Farrelly et al., 1999). TheECM is proposed to act through a6b1-integrin receptors toaffect cell survival, partially by stabilising PI-3 kinase-dependent survival responses to insulin (or IGF-1) (Boudreauet al., 1995; Pullan et al., 1996; Delcommenne et al., 1998;Farrelly et al., 1999). The Erk 1/Erk 2 pathway has alsobeen shown to be essential for laminin-rich ECM stimulatedsurvival of mammary epithelial cells. Thus, ECM-integrin me-diated intracellular signalling, through the formation of focaladhesions and subsequent downstream signalling, regulatesa number of key physiological responses in PMMEC. Assuch fundamental roles are attributed to the ECM, we soughtto delineate which genes are induced and repressed in theabsence of an overlay with particular emphasis on the formergroup as these genes are correlated with an undifferentiatedand apoptosing cell population.

A number of genes, whose expression is modulated byECM, have already been described. It has been shown thatthe ECM constituent laminin, while in the presence of lacto-genic hormones (in particular prolactin), drives the expressionof the b-casein gene in mammary epithelial cells (Streuli et al.,1995). Interleukin-1b converting enzyme (ICE) is a known

Published by Elsevier Ltd. All rights reserved.

473T. Lambe et al. / Cell Biology International 30 (2006) 472e479

inducer of apoptosis in mammalian cells, the level of mRNAexpression is down-regulated in the presence of ECM in mam-mary epithelial cells (Boudreau et al., 1995), as is the expres-sion of the c-myc, cyclin D1 and clusterin genes (Boudreauet al., 1996; Pullan et al., 1996). BRCA1 has been detected,through RT-PCR analysis, in PMMEC cultured in the absenceof ECM, but could not be detected in ECM overlaid cells(O’Connell and Martin, 2000). Transcript levels of Hoxb-7,Hoxa-1 and various integrin subunits are modulated in thepresence of ECM (Srebrow et al., 1998; Delcommenne andStreuli, 1995).

Previous studies examining gene expression patternsin mammary epithelial cells have largely been conducted ina hypothesis-driven manner. At present, there are a numberof techniques available to identify differentially expressedgenes in a less biased approach. Microarray technology allowsthousands of genes to be simultaneously profiled, while sup-pression subtractive hybridization (SSH) allows the directcomparison of two experimental populations (Diatchenkoet al., 1996). SSH employs critical normalisation steps allow-ing the creation of subtracted cDNA libraries for the identifi-cation of genes differentially expressed in response to anexperimental stimulus (Gurskaya et al., 1996). We show herethat expression of connexin 43 is associated, in vitro, withmammary cells cultured under proliferative or mildly apopto-tic conditions while, in vivo, high levels of expression are as-sociated with puberty, early pregnancy and early involution.

2. Materials and methods

2.1. RNA isolation, northern analysis and real-timePCR analysis

Total RNA was isolated from PMMEC and from mammary gland tissue

using TRIzol� Reagent (GibcoBrl, Life Technologies�). Northern blots

and SSH were performed as previously described (Murphy et al., 1999) and quan-

titated using either a phosphorimager (Bio-Rad) or with Scion Image� software.

For quantitative real-time PCR analysis, 1e2 mg of total RNA was DNase 1

treated and reverse transcribed using random primers and SuperScript� II (Gib-

coBrl). To 50e125 ng of cDNA template, 2X TaqMan Universal PCR Master

Mix, 300 nM of forward and reverse primers (connexin 43 fwd: AGA AGC

GAT CCT TAC CAC G and connexin 43 rev: GGA GAT CCG CAG TCT

TTG GT) and 150 nM Cx43 probe (CCA CCG GCC CAC TGA GCC C) were

added and made up to a final volume of 25 ml. Reactions were amplified and

quantified in an AB 7700 sequence detector using the supplied software (Applied

Biosystems). Cycling conditions were as follows: step 1, 2 min at 50 �C; step 2,

10 min at 95 �C; step 3, 15 s at 95 �C; step 4, 1 min at 60 �C; step 5, repeat from

step 3 for an additional 39 times. Experimental sample levels were normalised to

endogenous 18S rRNA levels (measured using control reagents and Pre-

developed Assay Reagent (PDAR) for 18S (Applied Biosystems)). To quantify

the relative level of connexin 43 the standard curve method was used: connexin

43 mRNA levels were quantified in a pool of RNA, including five serial dilutions,

and a standard curve of the serial dilutions versus Ct was plotted. Relative levels

of expression, in experimental samples, with respect to þECM samples were

then calculated and are expressed as fold increase or decrease.

2.2. Immunefluorescence analysis

Mammary glands were OCT embedded (tissue medium (Lab-Tek�)) and

5 mm thick sections were cut and fixed by immersion for 10 min in 1:1 meth-

anol:acetone at �20 �C. The sections were then washed in PBS and were used

directly or stored at �20 �C until they were processed. PMMEC were

harvested and cultured, as described below, in 2 well chamber slides

(Lab-Tek�) they were washed once in 1X PBS and then fixed in 4% parafor-

maldehyde for 20 min. Cells were permeabilised for 10 min with 1X TBST

containing 0.1% Triton X-100 and blocked with 5% normal goat serum in

1X TBST for 1 h, at room temperature. Primary antibodies, anti-connexin 43

(Signal Transduction Labs) 1/125 dilution, anti-smooth muscle actin (Sigma)

1/250 dilution, in 5% BSA and 5% normal goat serum in 1X TBST were incu-

bated overnight at 4 �C. Unbound antibody was removed by three 10 min

washes in 1X TBST. Incubation with fluorescent conjugated secondary anti-

body (FITC or TRITC conjugated, at 1/500 final dilution (Dako, Cambridge,

UK) in 5% BSA) was carried out for 1e1.5 h, at 37 �C. Nuclei were counter

stained with DAPI (5 mM) in TBST for 2 min and the images were captured

using Axioplan 2 Zeiss or Leica Leitz DMRB fluorescence microscope.

2.3. Cell culture

2.3.1. All treatments

Primary mammary epithelial cell cultures were prepared from 14- to 18-

day pregnant CD-1 mice as described by Barcellos-Hoff et al. (1989), Streuli

et al. (1991, 1995) and Furlong et al. (1996). The epithelial cells were resus-

pended in F12 medium containing gentamicin (50 mg/ml) and 10% foetal calf

serum (FCS) with the following hormones; hydrocortisone (H) 1 mg/ml

(Sigma, stock solution: 1 mg/ml in 100% ethanol), insulin (I) 5 mg/ml (Sigma,

stock solution: 5 mg/ml in 5 mM HCl), epidermal growth factor (EGF) 5 ng/

ml (Promega, stock solution: 5 mg/ml in F12 medium) and fetuin (Sigma,

1 mg/ml). Cells were seeded on tissue culture plastic at a density of

2.4� 106 cells/ml and grown for 48 h in this proliferation medium, cultured

at 37 �C in a humidified atmosphere of 5% CO2.

2.3.2. EGF samples

The cells were then harvested by scraping into Trizol�, RNA was extracted

immediately or the Trizol� suspension was stored at �80 �C prior to

extraction.

2.3.3. Kinase samples

Cultures were washed out and ‘‘starved’’ of EGF and serum, in the pres-

ence of hydrocortisone and insulin, for 6 h in F12 medium (with gentamicin).

An ECM matrix (Matrigel�, Becton Dickinson, 280 mg/ml, 2% v/v) diluted in

the above medium, in the presence or absence of the signalling pathway inhib-

itors, PD 98059 10 mM and SB 203580; 1 mM was added. After 36 h, the cells

were harvested as described above.

2.3.4. �ECM (& -I) samplesThe cells were washed and then cultured for a further 48 h in F12 medium

(with gentamicin) in the presence of 10% serum, EGF, insulin and hydrocor-

tisone. Cultures were washed out and ‘‘starved’’ of EGF and serum, in the

presence of hydrocortisone and in the presence or absence of insulin, for

24 (or 48 h) in F12 medium. The cells were harvested as described above.

2.3.5. þECM samples

The cells were washed and then cultured for a further 48 h in F12 medium

(with gentamicin) in the presence of 10% serum, EGF, insulin and hydrocor-

tisone. Cultures were washed out and ‘‘starved’’ of EGF and serum, in the

presence of hydrocortisone and insulin, for 24 h. Cultures were subsequently

washed and the medium changed: ECM matrix (Matrigel�, Becton Dickinson,

280 mg/ml, 2% v/v) diluted with F12, in the presence of hydrocortisone and

insulin. After 24 h, the cells were harvested as described above.

3. Results

3.1. Differential gene expression identified through SSH

SSH analysis suggested the differential induction of a lim-ited number of mRNAs, which were verified through northern

474 T. Lambe et al. / Cell Biology International 30 (2006) 472e479

blot analysis (Fig. 1). A focus of our study was the identifica-tion of genes associated with cells cultured in the absence ofan ECM as they are pre-disposed to apoptose. Thus, we fo-cused on the identification of these transcripts.

A moesin homologue was found to be up-regulated in cellscultured in the absence of ECM (Fig. 1A). Moesin is a memberof the Ezrin, Radixin, and Moesin (ERM) family of proteins.These proteins have been shown to be targeted at an earlystage of Fas ligand induced apoptosis, which is secondary toICE activation (Kondo et al., 1997). Fas and FasL expression

Fig. 1. Differential gene expression identified through SSH. Shown are repre-

sentative northern blot analyses for moesin homolog, eIF 3 and connexin 43.

At least three sets of RNA from independent cell culture preparations were

used. To normalise for loading the blots were stripped and re-probed with

an 18S ribosomal RNA probe and analysed by phosphorimaging. The left

panel of figures shows representative northern blots of tRNA from cells cul-

tured as described while the right panel graphically displays the levels of nor-

malised relative expression. The level of expression measured in the þECM

culture was set to 1 and all other values expressed relative to this; error bars

indicate standard error of the mean. (A) Lane 1, �ECM 24 h; lane 2,

þECM 24 h probed with 32P labelled moesin homolog cDNA. Normalised rel-

ative expression: 3:1. (B) Lane 1, �ECM 24 h; lane 2, þECM 24 h probed

with 32P labelled eIF 3 cDNA. Normalised relative expression: 1.6:1. No error

bars are displayed, as a pooled sample of three independent cultures was as-

sayed. (C) Lane 1, �ECM 24 h; Lane 2, þECM 24 h probed with 32P labelled

connexin 43 cDNA. Normalised relative expression: 2.2:1.

converge during involution of the mammary gland. In addi-tion, ICE protein accumulates during involution and activityhas been shown to be induced during apoptosis of culturedmammary epithelial cells (Boudreau et al., 1995; Martiet al., 2001; Song et al., 2000). ICE has also been shown to di-rectly induce a permeability transition in the mitochondriamarking a point of no return in the apopotic cascade (Susinet al., 1997). Thus, the expression of a moesin homologuemay be induced in vitro by ICE mediated apoptosis. A subunitof the eukaryotic translation initiation factor 3 (eIF 3), whichis a key determinant in the initiation of translation was foundto be up-regulated in cells cultured in the absence of ECM(Fig. 1B). Finally, connexin 43 was shown to be preferentiallyexpressed in cells cultured in the absence of ECM (Fig. 1C).

3.2. Connexin 43 mRNA expression in PMMEC

The differential expression of connexin 43, as identifiedthrough SSH, was robust and significantly different to warrantfurther study of this transcript. Cell culture of PMMEC wasused to disseminate the major signalling pathways crucialfor the expression of connexin 43 mRNA in vitro. Thus,PMMEC were cultured under a number of mildly apoptotic/quiescent and proliferative conditions and the expression levelof connexin 43 mRNA was determined and compared tothat of partially differentiated PMMEC. When compared toPMMEC cultured in the presence of an ECM overlay(þECM 24 h), connexin 43 mRNA expression was signifi-cantly higher in PMMECs, cultured in (1) the absence of anECM overlay (�ECM 24 h), and (2) in the presence of a strongproliferative drive mediated through EGF and FCS (þEGF/FCS) (Fig. 2A (lanes 3 and 4)).

As connexin 43 expression was linked with apoptotic stim-uli (�ECM overlay), we attempted to reverse the repressionseen in the þECM cultures by inhibiting the ECM mediatedpro-survival MAPK signalling pathway through the additionof the kinase inhibitors PD 98059 and SB 203580. However,this treatment did not change the levels of connexin 43mRNA significantly (Fig. 2B (lane 2)). Other strongly apopto-tic culturing conditions did not induce high levels of connexin43 mRNA expression (Fig. 2C (lane 1) and A (lane 1)). Thus,maximal connexin 43 expression was observed in cultureswhich contained large numbers of undifferentiated PMMEC(Fig. 2A (lanes 3 and 4). While minimal expression was cor-related with cells which had been differentiated in the pres-ence of an ECM or were actively apoptosing (Fig. 1C (lane2), Fig. 2A (lane 1), B (lane 2); C (lane 1)).

3.3. Connexin 43 protein expression in PMMEC

Connexin 43 protein in þEGF/FCS PMMEC was predom-inantly particulate and dispersed throughout the cell cytoplasmwith some staining at cellecell boundaries (Fig. 3A, bottomright-hand panel). Expression was not uniform in all cells.In contrast only ordered particulate connexin 43 staining atcellecell boundaries or at the cell membrane of individualcells was observed in the �ECM PMMEC (Fig. 3A, left-hand

475T. Lambe et al. / Cell Biology International 30 (2006) 472e479

Fig. 2. Expression of connexin 43 in PMMEC cultures. 32P labelled connexin 43 cDNA was used to probe tRNA from PMMEC. To normalise for loading the blots

were either (1) stripped and re-probed with an 18S ribosomal RNA probe and analysed by phosphorimaging or (2) total RNA levels were deduced using Scion

Image� analysis. The left panel of figures shows representative northern blots of tRNA from cells cultured as described while the right panel graphically displays

the levels of normalised relative expression. The level of expression measured in the þECM culture was set to 1 and all other values expressed relative to this; error

bars indicate standard error of the mean. (A) Lane 1, �ECM 48 h; lane 2, þECM 24 h; lane 3, �ECM 24 h; lane 4, �ECM þEGF. (B) Lane 1, �ECM 24 h; lane

2, þECM þPD 98059 (10 mM) þSB 203580 (1.0 mM) 30 h; lane 3, þECM 24 h. (C) Lane 1, �ECM �insulin 24 h; lane 2, �ECM 24 h; lane 3, þECM 24 h.

panels). This punctuate cell-membrane associated distributionmost likely reflects the organisation of connexin 43 into func-tioning connexons; again, connexin 43 expression was notseen in all cells. Fluorescence was not detected when the pri-mary antibody was omitted from the processing (data notshown). Cells cultured in the presence of an ECM overlaydid not show any connexin 43 staining (Fig. 3B).

Our enriched epithelial cell preparation may containa small number of myoepithelial cells and possibly stem cells(Barcellos-Hoff et al., 1989; Streuli and Bissell, 1990; Streuliet al., 1991, 1995; Furlong et al., 1996). To determine whichcells predominately express connexin 43 protein, we co-stainedthe cells with smooth muscle actin which stains myoepithelialcells (Dulbecco et al., 1986). Sporadic positive staining wasseen for smooth muscle actin in the cell preparations (Fig. 3A,

top right-hand panel and data not shown). In the �ECMPMMEC connexin 43 was seen on the cell peripheries of bothmyoepithelial and epithelial cells (Fig. 3A, top right-hand paneland data not shown). However, not all smooth muscle actinpositive cells were connexin 43 positive.

3.4. Connexin 43 mRNA expression in the developingmouse mammary gland

We next quantified the expression levels of connexin 43 atvarious developmental stages of the mammary gland. The invitro results predict high levels of connexin 43 expression dur-ing those stages of mammary gland development associatedwith either apoptosis (puberty and early involution) ora high mitotic index (puberty and pregnancy).

476 T. Lambe et al. / Cell Biology International 30 (2006) 472e479

Fig. 3. Immunocytochemical analysis of connexin 43 in PMMEC cultures. (A) The panels show typical immunocytochemical staining patterns of connexin

43 in PMMEC. Left-hand panels; PMMEC from �ECM 24 h stained for connexin 43 (FITC-green) and nuclei (DAPI-blue). Top right-hand panel; PMMEC

from �ECM 24 h stained for connexin 43 (FITC-green), smooth muscle actin (TRITC-red) and nuclei (DAPI-blue). Bottom right-hand panel; PMMEC from

�ECM þEGF stained for connexin 43 (FITC-green) and nuclei (DAPI-blue). Scale bar¼ 10 mM. (B) Shown is a typical immunocytochemical staining pattern

of connexin 43 (FITC-green) and nuclei (DAPI-blue) in PMMEC from þECM 24 h. Scale bar¼ 10 mM.

Independent analysis of connexin 43 expression by north-ern blot analysis showed highest expression in the pubertalgland with relatively significant levels in pregnancy and inthe 2-day involuting gland (Fig. 4A, panels a, b and d (lane2), respectively). No connexin 43 mRNA expression was de-tected in the lactating gland (however, a reactive species of

different size was observed) (panel c, lane 2). This patternof expression was confirmed through real-time PCR analysis(Fig. 4B); highest relative connexin 43 mRNA expressionwas again seen in the pubertal gland (5-week virgin) (panela) with relatively high levels of expression seen during earlypregnancy and in 24-h and 48-h involuting gland (panels

477T. Lambe et al. / Cell Biology International 30 (2006) 472e479

Fig. 4. Differential expression of connexin 43 during post-natal mammary gland development. (A) Northern analysis: top panels: 32P labelled connexin 43 cDNA

was used to probe tRNA from mouse mammary glands. (a) Lane 1, 3-week virgin; lane 2, 6-week virgin. (b) Lane 1, 2-day pregnant; lane 2, 16-day pregnant. (c)

Lane 1, 6 h surrogate; lane 2, lactating. (d) Lane 1, 6 h involuting; lane 2, 2-day involuting. Bottom panels: the blots were stripped and re-probed for equal loading

with a ribosomal RNA probe. An arrowhead indicates the connexin 43 transcript band. (B) Real-time PCR: relative connexin 43 cDNA levels in tRNA from mam-

mary glands of (a) virgin animals: series 1 [:]; series 2 [-]; and series 3 [C]: 3- and 5-week virgin (labelled 1 and 2, respectively); (b) pregnant animals: series 1

[:]: 2-, 14- and 18-day pregnant and lactating (labelled 1, 3, 4 and 5, respectively); series 2 [-]: 6-, 10-, 14- and 18-day pregnant and lactating (labelled 1e5,

respectively); and series 3 [C]: 2-, 10-, 14- and 18-day pregnant and lactating (labelled 1e5, respectively); (c) involuting animals: series 1 [:]; and series 2 [-]:

lactating, 3-, 9-, 24- and 48-h involuting (labelled 1e5, respectively); (d) re-suckled animals: series 1 [:]: 3-, 9- and 18-h re-suckled and lactating (labelled 1e4,

respectively); series 2 [-]: 3- and 18-h re-suckled and lactating (labelled 1, 3 and 4, respectively); series 3 [C]: 3-, 9- and 24-h re-suckled and lactating (labelled

1e4, respectively). Values are expressed relative to cDNA amount in lactation [set at 1.0] and presented graphically using a log scale.

b and c). By contrast, fully differentiated tissue, ie. lactatingand re-suckled mammary gland, expressed the lowest relativelevels of connexin 43 mRNA (Fig. 4B, panel d).

3.5. Connexin 43 protein expression in the developingmouse mammary gland

Connexin 43 expression in 2-day post-lactational involutingtissue was exclusively confined to a narrow cell layer at the

periphery of the alveoli (Fig. 5A, left-hand panel), localisingit to the myoepithelial cell population (Pitelka, 1988). Con-nexin 43 expression was not detected in lactating tissue(Fig. 5A, right-hand panel).

4. Discussion

The focus of our SSH study was the identification of genesassociated with PMMEC pre-disposed to apoptose. A limited

478 T. Lambe et al. / Cell Biology International 30 (2006) 472e479

Fig. 5. Immunocytochemical analysis of connexin 43 in the mouse mammary gland. (A) Shown are typical images of immunohistochemical staining of connexin

43 (FITC-intense green) of 5 mM thick sections of OCT embedded 2-day involuting tissue (left panel) and lactating tissue (right panel). Scale bar¼ 10 mM.

number of transcripts were identified. As the differential ex-pression of connexin 43 was reproducible and robust, furtherstudy of this gene, in mammary cells, was warranted.

Gap junctions form channels which connect neighbouringcells allowing the exchange of ions, metabolites and othersmall molecules, between the cytoplasm of adjacent cells(Beyer, 1993; Wolburg and Rohlmann, 1995). This facilitatesco-ordinating activities such as proliferation and differentia-tion (reviewed by Lo, 1999). Gap junctions have been pro-posed to play a number of key roles in physiologicalprocesses crucial for the development of the mammary gland(reviewed by Lo, 1999; Trosko et al., 1990). They are com-posed of connexons which are achieved through the oligomer-isation of six connexin molecules. Connexin 43 was shownhere to be preferentially expressed in PMMEC cultured inthe absence of ECM.

Connexin 43 mRNA expression has previously been show tobe expressed in a limited number of developmental stages of themammary gland where its expression has been suggested to al-low the passage of calcium between myoepithelial cells and thusbe crucial for the milk ‘‘let-down’’ from alveolus lumen to ductsduring lactation (Pozzi et al., 1995; Monaghan and Moss, 1996;Yamanaka et al., 1997). However, this suggested role for con-nexin 43 does not explain the differential expression reportedhere which was quantitatively associated with times of signifi-cant glandular remodeling. Alternatively, gap junctions com-posed of connexin 43 may function to facilitate glandularremodeling by allowing the passage of ‘‘cell fate’’ determiningmolecules. A role which has previously been attributed to con-nexin 43 facilitated intercellular communication, e.g. the ‘‘by-stander effect’’ where essentially non-apoptotic cells receiveapoptotic cues from their neighbours through gap junctions(Sanson et al., 2002; Azzam et al., 2001).

The finding that the ECM represses the level of connexin 43mRNA and that fully differentiated tissue, lactating andre-suckled mammary gland, expressed the lowest relative levelsof connexin 43 mRNA may indicate that terminally differenti-ated mammary cells do not express connexin 43. In support ofthis supposition is the proposed regulatory role for connexin43 mediated gap junction communication in early apoptosisand mitosis in the WB-F344 cell line (Wilson et al., 2000) andthe proposed necessity for gap junctional communication during

the differentiation of bovine mammary gland stem cells(Holland et al., 2003). Thus, connexin 43 mRNA expressionwas correlated with those developmental stages undergoingsignificant glandular remodeling while minimal expression isseen in the terminally differentiated gland suggesting that con-nexin 43 mediated gap junction communication may be playinghereto undiscovered roles in mammary gland development.

Previous studies of the 2-day post-lactational involutinggland have localised connexin 43 protein expression to themyoepithelial cell population in agreement with the data pre-sented here (Pozzi et al., 1995; Yamanaka et al., 1997). How-ever, connexin 43 protein has also been shown to be expressedin stromal and epithelial mammary cells, in a number of devel-opmental stages (Perez-Armendariz et al., 1995). The expres-sion of connexin 43 in a predominantly epithelial cell culturemay appear contradictory to the in vivo data. However, in vivostaining of connexin 43 in myoepithelial cells, which could ex-press relatively higher levels or different phosphorylatedforms, may mask punctuate staining in epithelial cells. Thus,cultures of PMMEC may make the detection of connexin 43protein in epithelial cells more feasible.

Alternatively, the culture of these epithelial cells may have in-duced a change in their phenotype, and so, their gene expressionpattern. The connexin 43 gene contains an IRES sequence andsuch sequences are generally found within genes whose transla-tion is maintained under stressful conditions (Schiavi et al.,1999). The absence of the myoepithelial sheath invitro may forcethe expression of this critical protein in epithelial cells.

This study shows that connexin 43 expression in the mam-mary gland is temporally restricted and suggests that expres-sion of this gene may be of importance in the post-natalcycle of mammary gland development. Resolution of the envi-ronmental and intracellular events that trigger connexin 43 ex-pression will be of interest. The discovery of the signals thattraverse connexin 43 gap junctions at these times, and theirdownstream targets is also of critical importance.

Acknowledgement

This work was supported by grants from the HealthResearch Board, Ireland, IRCSET and Science Foundation,Ireland.

479T. Lambe et al. / Cell Biology International 30 (2006) 472e479

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