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during mammary gland remodeling1Departamento de Bioquímica y Biología Molecular, 2Departamento de Medicina, and 3Unidad Central de Investigación, Facultad de Medicina-Fundación Investigación Hospital Clínico Valencia, Universidad de Valencia, Valencia, Spain
Submitted 4 September 2006 ; accepted in final form 1 December 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, RAR
, RAR
, and RXR, which underwent an increase in protein levels during weaning. In an attempt to elucidate the RAR-dependent signaling pathway, ChIP assays were performed. The results showed the binding of RAR to the MMP-9 promoter after 24- and 72-h weaning together with its coactivator p300; this fact could be responsible for the increase found in MMP-9 mRNA and protein levels in these conditions. Expression of related MMPs (MMP-2 and MMP-3) was also increased during weaning. Using gelatine zymography, we observed a time-dependent increase in active forms of MMP-9 and MMP-2. On the other hand, the inhibitor of MMPs, TIMP-1, was almost undetectable at 24- and 72-h weaning by Western blot. The role of retinoids in matrix remodeling is reinforced by the fact that administration of an acute dose of retinol palmitate to control lactating rats also induces MMP-9 expression. This emphasizes the importance of retinoids in vivo to regulate mammary gland involution.
mammary gland involution; matrix metalloproteinase-9; stromelysin-1; retinoic acid; retinoid acid receptor-
Few adult tissues exhibit extensive apoptosis under physiological conditions. These include the small intestine, adipose, uterus, ovary, and mammary gland (1, 9). After being weaned, the mammary gland is remodeled in preparation for the next pregnancy/lactation cycle through a complex cellular program. This weaning process has two phases. The first phase is reversible; it depends on p53 and is characterized by the disappearance of the physiological adaptations and by an increase in the number of apoptotic events in the epithelia of the lobulo-alveolar compartment (15, 33). The second phase is p53 independent; it is irreversible and includes a proteolytic degradation of the basement membrane and remodeling of the mammary gland. To achieve the latter events, two main families of extracellular matrix (ECM)-degrading proteinases are activated, matrix metalloproteinases (MMPs) and serine proteinases, that are involved in the activation of plasminogen to plasmin (11, 17). Recently, a third phase consisting of adipogenesis or biosynthesis has been ascribed to the regressing mammary gland (12).
MMPs are a broad family of more than 20 enzymes that are considered the key enzymes in the turnover of the ECM. Depending on their substrate specificity and domain similarity, MMPs are classified into collagenases, stromelysins, gelatinases, and membrane-bound MMPs (4, 22). The expression of MMPs is tightly regulated at the transcriptional and posttranscriptional levels by hormones, cytokines, and growth factors. Moreover, the activities of MMPs are also regulated by the presence of their inhibitors, tissue inhibitors of MMPs (TIMPs), and activators (plasmin, stromelysin, or MMP-2). Thus, a balance between MMPs and TIMPs regulates epithelial cell function and morphology during mammary gland development. During lactation, MMP activities remain low, since an intact basement membrane is required for the secretory phenotype; however, MMPs are highly expressed after being weaned, when there is a remodeling process essential for postweaning involution of the mammary gland (14, 16).
Retinoic acid (RA) and its precursor retinol (vitamin A) are known to be involved in the maintenance, differentiation, and function of many epithelial tissues. The pleiotropic effects of these compounds are mediated by nuclear retinoid receptors, which have the potential to transduce the RA signal in vivo. This family of receptors includes the retinoid acid receptors (RARs), which bind both all-trans- and 9-cis-RA steroisomers, and the retinoid X receptors (RXRs), which bind 9-cis-RA only. Each family is composed of three isotypes, designated , , and , encoded in separated genes. RARs and RXRs can form hetero- and homodimers that act as ligand-dependent transcription factors by binding to retinoid response elements in the promoters of RA target genes (7, 20, 21). In addition to the nuclear receptors, there are two cytoplasmic proteins involved in retinoid signal transduction, cellular RA-binding protein (CRABP I and CRABP II) and cellular retinol-binding protein (CRBP). All of them play an important role in retinoid homeostasis; CRBP acts to chaperone retinol- and retinal-to-retinoid-metabolizing enzymes, and CRABP II is thought to regulate the access of RA to nuclear receptors (8), whereas CRABP I is instead believed to be involved in RA catabolism (26).
Retinoids have been shown to modulate production of MMPs and TIMPs in mammary epithelial cells (24), endothelial cells, and alveolar macrophages (6, 10). Numerous studies (23, 32) in cell lines regarding the role of retinoids in breast cancer have been published. In vivo studies have shown that RA is required for proper morphogenesis of the mammary gland (30); however, little is known of the physiological role of RA in mammary gland involution. Here, we report the overexpression of CRABP II and RAR during weaning, which suggests that RA is involved in the involution of this tissue. Additionally, to highlight the role of retinoids on ECM remodeling, treatment of lactating rats with retinol palmitate induced transient increases of plasma retinoid levels and generated a slight decrease in milk production together with an induction of MMP-9 mRNA via RAR.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3 rats for each condition were used)], and another group that included rats at the peak of lactation that had been weaned for 24 h plus resuckling for another 24 h (n = 3). To determine the role of retinoids in mammary gland involution, four rats at the peak of lactation received a peritoneal injection of vitamin A (200,000 IU all-trans-retinol palmitate/kg body wt) 8 h before the experiment was performed. The trans-retinol palmitate (type VII, synthetic; Sigma-Aldrich, St. Louis, MO) was dissolved in 0.15 M NaCl. The control rats for the last group (n = 3) were injected with vehicle.
The rats were anesthetized with pentobarbital sodium (60 mg/kg body wt in 0.9% NaCl ip; Braun Medical, Rubi, Spain), and blood was collected from the aorta in heparinized syringes. Then, the inguinal mammary glands were removed and processed immediately.
Milk production. Milk production was measured as previously described (33). Briefly, the following equation relating pup milk yield to pup weight and weight gain was used to estimate milk production: yield = 0.0322 + 0.0667 (weight) + 0.877 (gain), where yield is daily yield per pup (g·day–1·pup–1), weight is pup weight (g), and gain is daily pup weight gain (g/day).
Retinoid determination. Retinol and retinol palmitate were measured in serum according to the method described by Barua and Olson (3).
RNA extraction and PCR analysis. Total RNA from mammary tissue was extracted using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). RT-PCR was performed as described previously (34), using the SuperScript reverse transcriptase (Invitrogen) and the Biotools DNA polymerase gel form (B&M Laboratories, Madrid, Spain). CRABP II, CRBP-1, and MMP-2, -3, and -9 mRNA expression were studied by RT-PCR using specific oligonucleotide primers (Table 1). Products were separated by agarose gels (1.2% wt/vol) and stained with ethidium bromide.
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(CT), where CT = CT target – CT housekeeping, and (CT) = CT treated – CT control. Chromatin immunoprecipitation assays. Chromatin from mammary tissue was fixed and immunoprecipitated according to Borrás et al. (5). Briefly, mammary tissue samples were excised and immersed in PBS, pH 7.4, and 1% formaldehyde for 8 min to crosslink the transcription factors to DNA. The reactions were stopped by adding glycine to a final concentration of 0.125 M. After centrifugation at 1500 g for 5 min, the cell pellet was resuspended in cell lysis buffer (85 mM KCl, 0.5% NP-40, 5 mM HEPES, pH 8.0) supplemented with a protease inhibitor cocktail (Sigma-Aldrich), incubated on ice for 15 min, and centrifuged at 3500 g for 5 min to pellet the nuclei. The pellet was resuspended in nuclear lysis buffer (10 mM EDTA, 1% SDS, 50 mM Tris·HCl, pH 8.1) at a ratio 1:1 (vol/wt) relative to the initial tissue weight, incubated on ice for 10 min, aliquoted in 1-ml fractions, and stored at –80°C until use for chromatin immunoprecipitation (ChIP) assay.
Cross-linked chromatin (1 ml of each sample) was sonicated on ice with 10 pulses of 10 s and 38% amplitude in a Vibra-Cell VCX-500 sonicator. The average chromatin size of the fragments obtained was 
500 bp. The sonified chromatin was treated as described previously (34). The antibodies used in this case were p300 and RAR (both from Santa Cruz Biotechnology, Santa Cruz, CA).
PCR analysis of immunoprecipitated chromatin. After DNA purification, input, immunoprecipitated, and unrelated antibody fractions were analyzed by PCR with the appropriate primer pairs to amplify products of 180–300 bp in length, corresponding to either the promoter or coding regions of the target genes (see Table 1). PCR fragments were size fractionated by 2% agarose gel electrophoresis and stained with ethidium bromide.
Protein extraction and immunoblotting analysis. The freeze-clamped tissue (0.1 g) was homogenized in 1 ml of extraction buffer as described previously (33). The lysates were normalized for protein concentration using a Bradford-based assay (Bio-Rad protein assay). Equal amounts of protein (10 µg) were loaded in SDS-PAGE gels and analyzed by Western blot, as described previously (33). Antibodies for CRABP II and CRBP-1 were a generous gift from Dr. Cecile Rochette-Egly and Dr. Christine Chaponnier, respectively. The rest of antibodies were all from Santa Cruz Biotechnology. Equal loading was confirmed by tubulin expression and Coomasie staining.
Gel zymography. Gelatine zymography was carried out as described by Heussen and Dowdle (13). In brief, protein lysates (150 µg) were diluted in nonreducing SDS sample buffer (62.5 mM Tris·HCl, pH 6.8, 25% glycerol, 4% SDS, and 0.001% bromophenol blue) and separated on 10% SDS-PAGE minigels containing 1 mg/ml gelatin (Bio-Rad). After electrophoresis, the gels were washed for 30 min at room temperature in renaturing buffer (10 mM Tris·HCl, pH 7.5, 2.5% Triton X-100), incubated for 36 h at 37°C in enzyme buffer (50 mM Tris·HCl, pH 7.6, 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35), and stained with Coomasie Brilliant Blue R-250. Gelatinolytic activities were detected after destaining by clear bands, indicating the lysis of the substrate.
Statistics.
Data are presented as means ± SE. Statistical significance was evaluated with one-way ANOVA. The homogeneity of the variances was analyzed by the Levene test; in those cases in which the variances were unequal, the data were adequately transformed before ANOVA. The null hypothesis was accepted for all the values of these sets in which the F value was nonsignificant at P > 0.05. The data for which the F value was significant were examined by Tukey's test at P < 0.05. In 
Fig. 7, statistical analysis was performed by the least significant difference test. After the analysis of the variances, the sets of data in which F was significant were examined by the modified t-test, using P < 0.05 as the critical limit.
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RESULTS |
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When analyzing the levels of both cellular retinoid carriers, CRBP-1 and CRABP II, by Western blot, an increase in CRBP-1 and CRABP II levels was shown after 24- and 72-h weaning (Fig. 1B). These values returned to control levels after resuckling. The time and rate of increase in protein levels were consistent with the increase in mRNA assessed by real rime RT-PCR.
The effects of retinoids are mediated through the RAR signaling pathway.
Retinoids mediate their biological activity by binding to specific nuclear receptors. To identify the receptors involved in the regulation of mammary regression, we performed immunoblot analysis in mammary tissue of lactating and weaned rats. Within the RAR subfamily, RAR is the most ubiquitously expressed, whereas RAR and RAR display more restrictive patterns of distribution (7). As shown in Fig. 2, RAR and RAR protein levels were increased as early as 2-h weaning and remained elevated in involuting mammary glands. On the other hand, RAR protein levels increased after 8-h weaning, reaching a peak after 24 h and decreasing thereafter. When studying the RXR subfamily, it was demonstrated that RXR protein levels were also elevated during the weaning process (Fig. 2). Interestingly, this increase was reversed toward control values after resuckling.
Induction of MMP-9 after weaning.
An increase in MMP-9 (92 kDa gelatinase, gelatinase B) in mammary epithelial cells upon RA treatment, mediated through an RAR-dependent signaling pathway, has already been reported (24). To determine whether weaning modulates MMP-9 expression through the RAR signaling pathway, real-time RT-PCR was performed in the mammary tissue from lactating and weaned rats. As seen in Fig. 3A, the MMP-9 mRNA expression showed a twofold increase after 24-h weaning, and this increase was even more evident in rats weaned for 72 h. In accordance with the previous result, the protein levels for MMP-9 were increased after weaning in a time-dependent manner, reaching a plateau at 72-h weaning (Fig. 3B).
RAR induces MMP-9 mRNA expression after weaning.
To examine in vivo the potential role of RA on the expression of MMP-9, ChIP assays were performed with an affinity-purified antibody directed against RAR and the coactivator p300 (Fig. 4A). DNA was extracted from the input, bound, and unrelated antibody fractions; equal amounts from each fraction were amplified using primers specific for the MMP-9 promoter region. The binding was determined by the relative intensity of ethidium bromide fluorescence compared with the input control. Our data show that RAR binding to the MMP-9 gene was evident after 8-h weaning, reaching a maximum at 24 h, whereas it was almost undetectable during lactation. Regarding the RAR coactivator p300, it was bound to the MMP-9 gene at 8-, 24-, and 72-h weaning. The binding to the MMP-9 coding region, used as a negative control, was not observed (Fig. 4B), indicating a specific binding of these transcription factors exclusively to the MMP-9 promoter.
Induction of other MMPs in involuting mammary gland. MMPs are essential in the ECM remodeling that takes place during the involution of the mammary gland. Indeed, MMP-9 is a key proteinase governing the degradation of basement membrane collagen as well as different types of gelatine. In this context, we have also studied the temporal secretion profile of other ECM-degrading MMPs, MMP-2 (72 kDa gelatinase or gelatinase A) and MMP-3 (stromelysin-1), during weaning. Real-time RT-PCR analysis was performed in rats at the peak of lactation and in weaned rats (Fig. 5A). The results revealed that the amounts of MMP-2 and MMP-3 mRNAs were significantly higher after 24-h weaning than those of lactating rats. This induction is time dependent and seems to diminish after the resuckling of the litter.
To determine whether the higher expression of MMPs was correlated with protein levels, we performed Western blot studies (Fig. 5B). Evaluation of protein expression revealed that MMP-3 was significantly increased after 24-h weaning and remained high for longer periods. The same result was observed for MMP-2, although in this case a second band of 55 kDa, corresponding to the active form of the enzyme, appeared as early as 8-h weaning and was at maximum at 72 h. In contrast with the result obtained by RT-PCR, this protein increase was not reversed after resuckling for either MMP-2 or MMP-3.
Temporal activity of gelatinases A and B in mammary gland involution. MMPs are synthetized as latent zymogens, which have to be biologically activated by cleavage of their proenzyme peptides (29). Both pro-MMP and its active form are bound by TIMP-1, retarding activation and proteolytic activity of these MMPs. To determine whether the higher expression of MMPs was correlated with increased enzyme activity, we performed zymography studies. As seen in Fig. 6A, there was a significant increase in gelatinase activities (MMP-2 and/or MMP-9) after weaning. Activation of pro-MMP-2 (70-kDa band) to an active form occurred at 72-h weaning. The upper band (105 kDa) likely represents pro-MMP-9 and also appears after 72-h weaning. This increased enzyme activity correlates with mRNA and protein levels in the weaned mammary tissue (Figs. 3 and 5).
Protein analysis by Western blot revealed that the inhibitor TIMP-1 decreased during weaning and was almost undetectable after 72-h weaning (Fig. 6B), which explains the increase in MMP-2, -3, and -9 levels.
MMP-9 induction is dependent on retinoid increase.
To elucidate the physiological role of RA on MMP-9 induction, control lactating rats were treated with an acute dose of retinol palmitate over a period of 8 h. In these rats, the concentration of this compound, measured by HPLC, was significantly higher in arterial blood compared with controls (data not shown). The binding of RAR to MMP-9 promoter was studied using the immunoprecipitation of complex RAR-DNA with purified antibodies reactive to RAR and the coactivator p300. DNA was extracted from the input, bound, and unrelated antibody fractions; equal amounts from each fraction were analyzed using primers that amplify the MMP-9 promoter region. Our data show that the RAR and p300 binding to the MMP-9 gene was almost undetectable in control lactating rats injected with vehicle, whereas in acute retinol palmitate treatment the binding was significantly higher (Fig. 7A). The binding to the MMP-9 coding region, used as a negative control, was not observed (Fig. 7B), indicating a specific binding of these transcription factors exclusively to the MMP-9 promoter. Finally, real-time RT-PCR demonstrated that this RAR binding to MMP-9 led to a higher expression of this gene in rats treated with retinol palmitate (Fig. 7C).
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DISCUSSION |
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The mammary gland is a highly dynamic organ that undergoes growth and remodeling during pubertal development and lactation. Both systemic and local hormones regulate the development and morphogenesis of the mammary gland. At the end of lactation, in response to diminishing lactational stimuli and milk stasis, the mammary gland undergoes involution that involves extensive epithelial apoptosis and tissue remodeling (11, 12). This process consists of a primarily reversible phase of apoptosis, a secondary irreversible phase of apoptosis and tissue remodeling, and a tertiary phase of adipogenesis. These stages bring about the dissapearance of milk-secreting acini and the reversion of the gland to a virgin-like state. The first stage is considered proteinase independent and p53 dependent (15, 33). It entails milk accumulation, induction of genes involved in mediating apoptosis, and initial apoptosis of epithelial cells. During this early phase, there was a strong induction of p53 and other genes participating in cell cycle arrest and apoptosis (33). However, very low levels of MMPs were present in the gland, whereas TIMP-1 was upregulated, shifting the proteolytic balance in favor of inhibition. The second proteinase-dependent stage begins around day 3 of involution and is characterized by a decrease in lactogenic hormones, collapse of glandular structures, and basement membrane destruction, resulting in active tissue remodeling and irreversible loss of the differenciated functions of the mammary gland (9, 11, 12, 18). This phase is characterized by upregulation of mRNA and activity for proteolytic enzymes, including gelatinases A and B and stromelysin-1, and downregulation of the inhibitor TIMP-1 (18). The third stage, recently termed the biosynthetic stage, involves adipose differentiation and reconstitution. This process can be artificially achieved by forced removal of the pups.
In this context, we have found that the RA signaling pathway is fully activated during involution, assessed by the increased expression of retinoid-binding proteins and nuclear RA receptors. As previously mentioned, it has been demonstrated (8) that retinoids exert their effects through retinoid-binding proteins, facilitating the delivery of RA to RAR and thereby enhancing the transcriptional activity of the receptors. These observations suggest that upregulation of direct RAR target genes will be increased by CRABP II. As shown in Fig. 1, our results demonstrated that the retinoid carrier proteins CRABP II and CRBP-1 were sharply increased during weaning compared with lactating glands. Moreover, the mRNA levels for CRABP II were increased 10-fold after 72-h weaning (Fig. 1A), in good agreement with previously published Affymetrix data (34). Interestingly, we found that both mRNA and protein levels reverted to control values after resuckling, suggesting a reversible mechanism of action of retinoids at this phase of involution.
In parallel with these results, retinoid nuclear receptor (RAR, RAR, and RXR) protein levels were increased as early as during 2-h weaning, remained elevated in involuting mammary glands, and yet again reverted towards control values after resuckling (Fig. 2). On the other hand, RAR increase is delayed, reaching a sharp peak at 24 h and declining thereafter. Retinoid receptors have already been studied at different stages of mammary gland development; in this sense, it has been described that RAR is strongly expressed throughout mammary gland development (30), whereas RAR is expressed only at specific time points (30). It has been suggested that RAR may be responsible for RA signaling in nulliparous and pregnant mammary gland, whereas a combination of RAR and RAR may be required for RA signaling in lactating mammary gland. Our results suggest that, most likely, this could also state for mammary gland involution. Interestingly, although RAR protein levels are barely detected in virgin, pregnant, or lactating mammary gland (30), our results demonstrate that this protein is strongly induced during mammary gland involution. Taken together, these data suggest that different RA receptors function in distinct phases of the mammary gland development, assigning RAR a prominent role during mammary gland regression, since this is the only stage where RAR is expressed in mammary gland.
Regarding the biological significance of these findings, the prominent role played by RARs in the modulation of MMP expression has been demonstrated extensively (24, 30). Matrix-degrading proteinases clearly have an important function as mediators of the tissue remodeling that occurs during mammary gland involution. Previous work has detailed the expression of secreted MMPs in mammary gland development; MMP-2, -3, and -9 are present in the mammary tissue and play a role not only in branching morphogenesis (16, 31) but in involution as well (2, 18). Indeed, some of the mouse strains deficient for specific secreted MMPs display altered mammary tissue morphology or involution (31). However, few studies (28) have focused on the regulation of MMP expression during involution, being difficult to predict which factors induce the expression of MMPs. The data presented here show a relationship between RA and MMP-9 expression, suggesting an important role of retinoids in the development and involution of glandular epithelial structures. Using chromatin immunoprecipitation assay, we demonstrated for the first time that RAR, although absent from MMP-9 promoter in lactating controls, is recruited to the MMP-9 promoter in mammary gland during weaning (Fig. 4). Moreover, it has been shown that p300 acts as a necessary cofactor mediating nuclear receptor-activated gene transcription. Herein, we demonstrate p300 binding to the MMP-9 promoter. These data correlate with the transcription of the gene as seen by quantitative RT-PCR, in which the expression was increased severalfold after 24- and 72-h weaning (Fig. 3). Overall, these results strongly suggest that RAR and p300, as integrators of the signaling pathway, modulate the expression of MMP-9 during mammary gland involution.
ECM remodeling is a well-orchestrated process in which it is likely that a functional overlap exists between different MMPs. In our experimental conditions we have also seen an upregulation of other proteinases, such as MMP-3 and MMP-2, during the mammary gland remodeling (Fig. 5). However, in the case of these two MMPs, the mRNA and protein levels did not return to control values after the resuckling of the litter, as occurs with MMP-9. Previous publications (25, 30) have described MMP-3 to be downregulated by RAR, although the specific RAR involved in this modulation is not known. It has been suggested (25) that one of the members of the RAR family is competing with activating protein-1 for the DNA binding site. Possibly, as mentioned above, RARs function in a differential manner in distinct phases of the mammary gland development. Additional studies would be necessary to elucidate the precise mechanisms governing the different pattern of expression of MMP-3 by RAR throughout mammary gland evolution. On the other hand, the increase in MMP-2 expression, although not a target gene for RA, emphasizes the importance of a tight coordination between matrix-degrading enzymes, since it presents the same pattern of expression as MMP-3.
The regulation of proteases in the mammary gland can be achieved in three different ways: at the mRNA transcription level (1), at the zymogen activation level (2), and at the specific inhibitor level (15). Herein, we demonstrate that the higher expression of MMPs correlated with an increased enzymatic activity of MMP-2 and -9 after weaning (Fig. 6A). We also observed that TIMP-1, the inhibitor of MMPs, dramatically decreased after weaning and was almost undetectable after 72 h (Fig. 6B). This result could explain the increased activity observed in the MMPs studied in this work. In fact, mice overexpressing TIMP-1 do not display an involution phenotype (11). On the other hand, the importance of keeping an accurate MMP-to-TIMP ratio to achieve a correct tissue remodeling is well known; otherwise, an excessive matrix degradation or matrix deposition could lead to tumourigenesis or fibrosis.
It has been described (24) that RA induces lumen morphogenesis in mammary epithelial cells. This in vitro approach demonstrates that the production of MMP-9 in this cell line is induced, in a dose- and time-dependent manner, by the addition of RA via the RAR receptor. There are also studies in vivo in which RA is essential for the maintenance of the ECM in renal basement membranes (19) or even for the morphogenesis of the mammary gland (30). To corroborate the role of retinoids on the regulation of MMP-9, control lactating rats were treated with an acute dose of retinol-palmitate 8 h before death. This group of rats showed a significant decrease in milk production (results not shown), and, in parallel to the results found during weaning, there was a binding of RAR and p300 to the MMP-9 promoter, which was followed by the induction of the gene (Fig. 7).
These findings point out the importance of RA not only in the development and maintenance of alveolar epithelial structures (24, 30) but also in the remodeling of the gland that takes place at the end of lactation. Therefore, retinoids present a dual role in the gland that is essential for proper morphogenesis of the tissue and for the regression at the end of lactation. This fact points out the physiological role of retinoids as a potential signal that triggers mammary gland involution during weaning. Furthermore, it would be of great interest for the future to study the role of RA in the modulation of unconventional roles of MMPs and TIMPs, such as apoptosis or releasing growth factor attached to the ECM that could be of relevance in the mammary gland development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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B modulation in the lactating mammary gland: importance of GSH. Biochem J 391: 581–588, 2005.[CrossRef][ISI][Medline]This article has been cited by other articles:
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  P. D. Curtis, J. Atwood III, R. Orlando, and L. J. Shimkets Proteins Associated with the Myxococcus xanthus Extracellular Matrix J. Bacteriol., November 1, 2007; 189(21): 7634 - 7642. [Abstract] [Full Text] [PDF]
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