2Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, PO Box 3640, D-76021 Karlsruhe, Germany, and 3Division of Allergy, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA
Received on September 3, 2001; revised on October 26, 2001; accepted on November 2, 2001.
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Abstract |
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Key words: ABH histo-blood group antigen/apoptosis/galectin-3/mammary gland/tissue remodeling
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Introduction |
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The M-N1 antigen is virtually nonexpressed in normal mammary tissue but is strongly up-regulated during involution of these glands after the cessation of lactation (Mengwasser and Sleeman, 2001). However, the incidence of apoptotic bodies during tissue involution inversely correlates with M-N1 staining. Thus M-N1 expression may confer a growth or survival property onto those epithelial cells required for tissue remodeling during mammary gland involution. This finding is consistent with the notion that the expression of the M-N1 antigen promotes tumor cell growth or survival in vivo.
A possible explanation for the blocking of tumor growth by the M-N1 antibody is that it inhibits the interaction of tumor cells with ligands or extracellular matrix components vital for their growth or survival in vivo. A candidate for such a ligand would be galectin-3. Galectin-3 is an endogenous mammalian lectin that binds to ABH antigens (Sato and Hughes, 1992), and can be located in the cytoplasm, located in the nucleus, or secreted extracellularly (reviewed in Hughes, 1999
). A number of important biological functions have been ascribed to galectin-3, including modulation of cell adhesive properties (Inohara and Raz, 1995
; Kuwabara and Liu, 1996
; Warfield et al., 1997
; Ochieng et al., 1998
) and regulation of cell motility (Sano et al., 2000
). Additionally it has anti-apoptotic and growth-enhancing properties (Yang et al., 1996
; Akahani et al., 1997
) and can promote cell invasiveness (Le Marer and Hughes, 1996
). These abilities of galectin-3 are obviously closely tied with aspects of tumorigenesis and metastasis. Indeed, in several cases expression of galectin-3 has been shown to correlate with histological grade and to be prognostically significant (Nakamura et al., 1999
; Honjo et al., 2000
). More significantly, ectopic expression or inhibition of galectin-3 has been shown to increase or decrease tumorigenicity and metastatic proclivity of tumor cells, respectively (Bresalier et al., 1998
; Yoshii et al., 2001
; Honjo et al., 2001
).
Our aim in the study presented in this article was to examine the expression pattern of galectin-3 in the mammary gland to determine whether galectin-3 expression correlates with M-N1 antigen expression. Prior to pregnancy and lactation, mammary glands contain branching networks of ducts formed by mammary epithelial cells (reviewed in Vonderhaar, 1985). During pregnancy and lactation, lateral buds form along the ducts and subsequently develop into alveoli. These alveoli are made up of differentiated mammary epithelial cells that secrete milk during lactation. After weaning, the mammary glands regress in a process called involution. During involution the mammary gland is restructured through the coordinated processes of apoptosis of luminal epithelial, myoeptithelial, and endothelial cells, and lobular-alveolar remodeling (Pitelka, 1988
; Lund et al., 1996
; Strange et al., 1992
). Loss of suckling leads to accumulation of milk in the alvoeli and a fall in the levels of systemic lactogenic hormones. These events trigger the involution process (Feng et al., 1995
; Marti et al., 1997
).
There are two distinct phases to mammary gland involution. The first phase is reversible and controlled by local factors. Alveolar cells apoptose but no remodeling of the lobular-alveolar structure occurs (Li et al., 1997). In the second phase, proteases degrade extracellular matrix and basement membrane components and the lobular-alveolar structures collapse (Lund et al., 1996
). Continued apoptosis, replacement of most of the epithelial component with adipose tissue and reestablishment of the resting mammary gland ductal structures, leads to remodeling of the gland. The involution process is completed within 1015 days (Lascelles and Lee, 1978
).
Here we show that galectin-3 is specifically and strongly up-regulated during mammary gland involution, virtually exclusively on nonapoptosing mammary epithelial cells. There is considerable colocalization of galectin-3 and M-N1 expression. The increased expression of galectin-3 during mammary gland involution is regulated at the transcriptional and posttranscriptional levels and is sensitive to glucocorticoid hormones.
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Results |
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Discussion |
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Galectin-3 has a number of properties that could contribute to tissue remodeling, incuding the regulation of cell adhesiveness and motility, the suppression of apoptosis, and the promotion of cell growth and invasiveness. Furthermore, galectin-3 has been shown to modulate branching morphogenesis of the uretic bud/collecting duct in the embryonic kidney (Bullock et al., 2001), and we note that this branching morphogenesis has many similarities to the tissue remodeling that occurs in the postlactation mammary gland. Which of the properties of galectin-3 might contribute to tissue remodeling? We observed virtually exclusive expression of galectin-3 on nonapoptotic cells. Thus in the context of tissue remodeling, expression of galectin-3 might suppress the induction of apoptosis. Additionally, dexamethasone treatment inhibits tissue remodeling but not apoptosis in involuting mammary glands (Lund et al., 1996
; Li et al., 1997
; Mengwasser and Sleeman, 2001
; data presented herein) and also suppresses galectin-3 expression (Figure 3, Figure 5, Table II). If galectin-3 were to suppress apoptosis in the involuting mammary gland, one prediction would be that dexamethasone treatment might result in an increased rate of apoptosis. We note that dexamethasone treatment results in a modest increase in the number of apoptotic cells in involuting mammary glands (Table II). However, it is equally possible that properties of galectin-3 other than or in addition to the suppression of apoptosis might contribute to tissue remodeling.
If galectin-3 plays a role in postlactational tissue remodeling, one might expect to see disturbed tissue remodeling in mice bearing a targeted deletion of the galectin-3 gene. We examined this histologically by comparing postlactational mammary glands from wild-type mice with those from galectin-3 knockout animals (Hsu et al., 2000), but could observe no obvious morphological or kinetic differences in the involution process (data not shown). However it is important to remember that there are many members of the galectin family, and it is highly likely that other members may compensate for the lack of galectin-3 in the knockout animals. Therefore it will be important to use conditional knockout techniques to examine the role of galectin-3 in mammary gland involution.
An unexpected observation in this study was the fact that galectin-3 protein levels are regulated differently to galectin-3 RNA levels, suggesting that galectin-3 is both transcriptionally and posttranscriptionally regulated. The glucocorticoid hormone dexamethasone appears to act posttranscriptionally because no difference in galectin-3 RNA levels was observed when involuting mammary glands from dexamethasone-treated and nontreated animals were compared. This would suggest that dexamethasone acts indirectly, perhaps by regulating galectin-3 translation or by affecting galectin-3 protein stability.
Such a scenario is not without precedent. For example, dexamethasone blocks the IL-4-induced increase in protein levels of IL-4Ralpha on T and B cells without altering IL-4Ralpha mRNA levels (Mozo et al., 1998). Glucocorticoids are known to negatively modulate protein synthesis, in part through inhibiting the assembly of the functional eIF4F holocomplex required for translation initiation (Shah et al., 2000a
,b), and through attenuation of the activation of ribosomal protein S6 kinase (Shah et al., 2000b
,c) whose role is to preferentially select those mRNA species bearing a cis-acting terminal oligopyrimidine (5'-TOP) motif for translation. Furthermore, glucocorticoids can also stimulate proteasome- and calcium-dependent proteolysis (Wang et al., 1998
; Thompson et al., 1999
). If our suggestion that galectin-3 plays a role in tissue remodeling is correct, then the suppression of galectin-3 protein expression by systemic glucocorticoid hormones during the first stage of involution (despite the high galectin-3 RNA levels present at this time) might be important for ensuring that tissue remodeling does not occur during this first, reversible stage.
Our initial motivation for analysing expression of galectin-3 in involuting mammary glands was to compare its expression with that of the M-N1 antigen, to determine the likelihood that this carbohydrate antigen might be a ligand for galectin-3 during involution. Like M-N1, galectin-3 is strongly up-regulated during involution and is also virtually never expressed in apoptotic cells. M-N1 and galectin-3 also colocalize (Table I). Thus galectin-3 and M-N1 are spatially and temporarily expressed such that M-N1 could act as a ligand for galectin-3. However, the colocalization of M-N1 and galectin-3 is by no means complete. Furthermore, M-N1 is not regulated by glucocorticoid hormones (Mengwasser and Sleeman, 2001). Future work will therefore focus on determining whether M-N1 and galectin-3 have a functional relationship.
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Materials and methods |
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Immunohistochemistry
Mammary glands were fixed in 4% paraformaldehyde and then embedded in paraffin wax and sectioned. The sections were subsequently immunostained as described (Dall et al., 1995), using 3,3-amino-9-ethyl carbazole for color development. The monoclonal antibodies used in this study were M-N1 (Sleeman et al., 1999
) A1D6 anti-galectin-3 (Liu et al., 1996
), and AC-15 anti-ß-actin (Sigma). For double-staining experiments, sections were stained first with galectin-3 or ß-actin antibodies using alkaline phosphatase standard (Vector Laboratories) and the alkaline phosphatase Substrate Kit III (blue color, Vector Laboratories) for color development. Subsequent staining of apoptotic cells was performed using an Apoptag® kit (Oncor) according to the manufacturers instructions, except that alkaline phosphate Substrate Kit I (red color, Vector Laboratories) was used for color development. Quantification of staining was performed by taking photographs of stained sections and counting the number of stained cells in five independent 1-mm square fields of view.
Cloning of rat galectin-3
Full-length rat galectin-3 was amplified by reverse-transcription polymerase chain reaction from rat spleen cDNA and cloned into the BamHI/EcoRI sites of pcDNA3.1. The primers used were 5'-GCGGA TCCAT GGCAG ACGGC TTCTC ACTTA ATGAT G-3' and 5'-GCGAA TTCTT AGATC ATGAT GGCGT GGGAA GCGCT GG-3'. The amplified sequence corresponds to bases 41830 of the published rat galectin-3 sequence (GenBank accession number NM031832). The amplified cDNA was multiply sequenced on both strands to ensure the sequence was correct. The galectin-3 cDNA was cut out of the pcDNA3-1 vector and used as a probe for northern blots.
Northern blots
RNA was prepared from snap-frozen mammary gland, colon, and stomach tissue using peqGOLD RNA Pure (Peqlab) according to the manufacturers instructions. Poly (A)+ RNA was subsequently purified from the total RNA using standard protocols, and 5-µg aliquots were size-fractionated on 1.0% formaldehyde-agarose gels and blotted onto Hybond N+ membrane (Amersham). The membranes were then cross-linked (UV Stratalinker 2400, Stratagene) and hydridized at 65°C in QuickHyb® (Stratagene). Probes were generated by 32P-labeling of cDNA fragments (ReadyPrime, Amersham). Unincorporated label was removed prior to hybridization using an Elutip (Schleicher & Schüll) according to the manufacturers specifications. After hybridization with the labeled probes, membranes were washed twice in 2x SSC, 0.1% sodium dodecyl sulfate (SDS), and twice in 1x SSC, 0.1% SDS at 64°C, after which they were exposed to film (20x SSC contains 175.3 g/l NaCl and 88.2 g/l sodium citrate, pH 7.0).
SDSPAGE and western blotting
Protein lysates from mammary glands were prepared as previously described (Hebbard et al., 2000). Briefly, freshly dissected mammary glands were snap-frozen in liquid nitrogen, then ground to a powder in liquid nitrogen using a pestle and mortar. Frozen powdered mammary gland was mixed with a preweighed vial of SDSpolyacrylamide gel electrophoresis (PAGE) sample buffer (2% SDS, 125 mM TrisHCl, pH 6.8, 100mM dithiothretol, 10% glycerol). The vial was then weighed again to determine to amount of frozen powdered mammary that had been added to the vial. The concentration was then adjusted to 25 mg frozen powdered mammary gland/ml sample buffer. The resulting protein lysate was sonified and then boiled for 5 min. Proteins were resolved by size by SDSPAGE (1 mg frozen powdered mammary gland per cm loading slot) using a resolving gel containing 10% polyacrylamide. For western blotting, proteins separated by SDSPAGE were electrically transferred to ImmobilonTM-P (Millipore) by the method of Towbin et al. (1979)
. Blots were probed first with A1D6 anti-galectin-3 (Liu et al., 1996
) antibody and subsequently with anti-ß-actin antibody clone AC-15 (Sigma) using the electrochemiluminescence detection system (Amersham, Braunschweig) as previously described (Sleeman, 1993
).
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Abbreviations |
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Acknowledgments |
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Footnotes |
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References |
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