Proteomic discovery of genistein action in the rat mammary gland

Genistein, the primary isoflavone component of soy, consumed in diet during the prepubertal period suppresses chemically induced mammary cancer in rats. The current study used two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology to identify proteins responsible for genistein breast cancer protection In Vivo. Female offspring were exposed via lactating dams treated with 250 mg genistein/kg AIN-76A diet from days 1 to 21 postpartum (prepubertal period). Mammary glands were collected at 21 and 50 day of age and subjected to 2-DE/MS and immuno-blot analyses. Twenty-three proteins were determined to be differentially regulated (p < 0.05) and identified using 2-DE, followed by MALDI-TOF/TOF or LC-ESI-MS/MS. Five of these proteins were validated by immuno-blots. Annexin A2 was significantly increased at 21 days yet found to be decreased at 50 days. Fetuin B was found to be unchanged at day 21 but increased at day 50. Phosphoglycerate kinase 1 (PGK1) was unchanged at day 21 but decreased at day 50. Gelsolin was increased at day 21 but not at day 50. Protein disulfide-isomerase A3 (PDIA3) was decreased at day 21 and unchanged at day 50. Also, we found that vascular endothelial growth factor receptor 2 (VEGF-R2) and epidermal growth factor receptor (EGF-R) were decreased in mammary glands of 50-day-old rats treated prepubertally with genistein. This study demonstrates the usefulness of proteomics for the discovery of key proteins involved in signaling pathways to understand genistein mechanisms of action in breast cancer prevention.     

Proteomic analysis in mammary glands of rat offspring exposed in utero to bisphenol A

Bisphenol A (BPA) is a ubiquitous environmental contaminant with established endocrine disruptor properties. The objective of our study was to determine the effects of prenatal exposure to BPA on the rat mammary gland proteome in postnatal rats as a first step toward the investigation of translational biomarkers of susceptibility in the human population. Pregnant rats were treated orally with 0, 25 or 250 µg BPA/kg body weight from days 10 to 21 post-conception. Female offspring were euthanized at 21 and 50 days, and mammary glands were collected. Proteomic analysis was conducted using 2-DE, followed by a combination of MALDI-TOF–TOF and LC–MS/MS, which led to the identification of 21 differentially abundant proteins including vimentin, SPARC and 14–3–3. Western blot analysis of key downstream signaling proteins demonstrated increased phospho-AKT, c-Raf, phospho-ERKs-1 and 2, but decreased TGF-β in mammary glands of 50 day old rats exposed prenatally to BPA. Our studies indicate for the first time that key proteins involved in signaling pathways such as cellular proliferation are regulated at the protein level by BPA. This data is expected to aid in the understanding of how BPA may be influencing the susceptibility of the mammary gland to cancer transformation.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Mammary gland morphogenesis is enhanced by exposure to flaxseed or its major lignan during suckling in rats

The exposure of rats to 10% flaxseed (FS) or an equivalent level of its major lignan, secoisolariciresinol diglucoside (SDG), during suckling enhances mammary gland differentiation, which protects against mammary carcinogenesis at adulthood. We determined whether this diet-induced mammary gland differentiation is mediated through the estrogenic pathway via epidermal growth factor receptor (EGFR) and estrogen receptor (ER) signaling. Rats were fed the AIN-93G basal diet (BD) from day 7 of pregnancy until delivery and then randomized to consume BD, FS, or SDG during lactation. After weaning, female offspring were fed BD throughout the experiment. At postnatal day (PND) 21 and the proestrus phase on PND 49-51, mammary glands of offspring were analyzed for morphology, cell proliferation, and expression of EGFR, epidermal growth factor (EGF), transforming growth factor-alpha, ER-alpha, and ER-beta. At PND 21, compared with the BD control, the number of terminal end buds (TEBs) and terminal ducts were increased by FS, whereas mammary epithelial cell proliferation was increased by both FS and SDG, suggesting that mammary morphogenesis was enhanced. Epithelial EGFR and stromal fibroblast EGF were increased by SDG, whereas epithelial ER-beta was decreased by FS. Conversely, at PND 49-51, a lower number of TEBs but a higher ratio of lobules to TEBs with decreased expression of EGFR or EGF was observed in both treatment groups. EGFR expression was positively associated with EGF expression and cell proliferation in TEB epithelium at PND 21. Urinary lignans of lactating dams were related to their offspring's indices of mammary gland development. In conclusion, exposure to FS or SDG during suckling enhanced mammary gland morphogenesis by modulation of EGFR and ER signaling, which led to more differentiated mammary glands at PND 49-51. The physiological outcomes of FS and SDG were similar, which suggests that SDG is partly responsible for the mammary gland differentiation effect.     

Acute involution in the tammar wallaby: identification of genes and putative novel milk proteins implicated in mammary gland function

Marsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution.     

Prenatal exposure to bisphenol A promotes angiogenesis and alters steroid-mediated responses in the mammary glands of cycling rats

Prenatal exposure to BPA disturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to chemical challenges administered long after BPA exposure. Our aim was to assess the effect of prenatal BPA exposure on mammary gland angiogenesis and steroid hormone pathways in virgin cycling rats. Pregnant Wistar rats were exposed to either 25 or 250 g/kg/day (25 and 250 BPA, respectively) or to vehicle. Female offspring were autopsied on postnatal day (PND) 50 or 110. Ovarian steroid serum levels, the expression of steroid receptors and their co-regulators SRC-3 and SMRT in the mammary gland, and angiogenesis were evaluated. At PND 50, all BPA-treated animals had lower serum levels of progesterone, while estradiol levels remained unchanged. The higher dose of BPA increased mammary ERα and decreased SRC-3 expression at PND 50 and PND 110. SMRT protein levels were similar among groups at PND 50, whereas at PND 110, animals exposed to 250 BPA showed a lower SMRT expression. Interestingly, in the control and 25 BPA groups, SMRT increased from PND 50 to PND 110. At PND 50, an increased vascular area associated with higher VEGF expression was observed in the 250 BPA-treated rats. At PND 110, the vascular area was still increased, but VEGF expression was similar to that of control rats. The present results demonstrate that prenatal exposure to BPA alters the endocrine environment of the mammary gland and its angiogenic process. Increased angiogenesis and altered steroid hormone signals could explain the higher frequency of pre-neoplastic lesions found later in life. This article is part of a Special Issue entitled 'Endocrine disruptors'.     

Variation of the poly(A) size classes in the rat mammary gland during the lactation cycle

The sizes of the poly(A) tracts associated with rat mammary RNA were determined at several time points in the lactation cycle. The poly(A) tracts in the lactating gland displayed two predominant size class peaks at 80-85 and 45-47 residues. The 9S whey protein mRNA and the 15S casein mRNA purified from the 12 day lactating mammary gland both contained poly(A) tracts displaying a similar size distribution. The 45 residue tracts were a characteristic of lactation; they were not found at 8 days of pregnancy and only small amounts of these shorter poly(A) tracts were found in the 16 day pregnant gland. The poly(A) tracts of the involuted gland displayed the same size characteristics as those of late pregnancy. At all the developmental stages that were examined, the fraction of 45 residue poly(A) tracts was always proportional to the total poly(A) content of the mammary cells.     

Prepartal lactose concentration in the mammary glands of Uje: WIST rats as a simple indicator of approaching parturition.

Immediately after delivery, lactose was reliably detectable in rat mammary glands (mean concentration 6.49 +/- 0.57 mg/g tissue). The concentrations were not influenced by prepartal biopsy of contralateral mammary tissue. In the morning of the 21st day of gestation (= expected day of labor), lactose was evident in the mammary glands of dams delivering at that day (mean concentration 2.39 +/- 0.17 mg/g tissue). In the tissue of dams delivering on day 22 of gestation, lactose was undetectable. A correlation between prepartal lactose concentration of mammary gland and time remaining up to beginning of delivery (r = -0.96) qualifies this parameter as a rough indicator of term of parturition.     

p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.     

Dietary regulation of mammary lipogenesis in lactating rats.

The proportion of medium-chain fatty acids (C8:0, C10:0 and C12:0) in rat milk increased significantly between day 4 and day 8 of lactation and for the remainder of lactation these acids comprised 40-50mol% of the total fatty acids. The milk fatty acid composition from day 8 was markedly dependent on the presence of dietary fat and altered to include the major fatty acids of the fats (peanut oil, coconut oil and linseed oil). The distribution of fatty acids made within the gland, however, was independent of dietary lipid and C8:0, C10:0 and C12:0 acids accounted for over 70% of the fatty acids made. The rates of lipogenesis in both the mammary gland and liver determined in vivo after the administration of 3H2O were affected by the presence of dietary lipid. In the mammary gland the rate for rats fed a diet containing peanut oil for 7 days was only one fifth that for rats fed a fat-free diet. Coconut oil also suppressed lipogenesis. Both dietary fats also suppressed lipogenesis in the liver.     

Effect of suboptimal nutrition during lactation on milk protein gene expression in the rat

Human milk provides infants with proteins that aid in the prevention of infections and facilitate the digestion and absorption of other nutrients. Maternal diet is not believed to affect the protein concentration of breast milk. However, the maternal factors that regulate the expression of genes for specific milk proteins are not well characterized. We hypothesized that nutrition could be one of the factors. We fed Sprague-Dawley rats five diets representing common nutrient deficiencies and energy deficiency during pregnancy and lactation: low-zinc (Zn; 7 microg/g), low-iron (Fe; 6 microg/g), low-protein (12.5% albumin), pair-fed control diet (lactation only, 20% less kcal) and control diet (Zn, 25 microg/g; Fe, 100 mug/g; protein, 21%) ad libitum. At day 10 of lactation, the mammary gland was removed for RNA extraction. Northern blots of mRNA from the different groups were performed by hybridization with beta-casein and whey acidic protein (WAP) cDNA probes. The expression of beta-casein mRNA in rat mammary gland was significantly (P<.005) increased in the pair-fed group when compared to the control group. The expression of WAP mRNA was also significantly (P<.005) increased in the pair-fed group as well as in the low-Fe group when compared to the control group. The concentration of beta-casein in milk was significantly higher for the low-zinc and the pair-fed groups only. The concentration of WAP in milk was not different among groups. These results suggest that compromised maternal nutrition can affect the expression of two individual milk proteins and may have functional implications with regard to bioactive proteins in milk.     

Leptin mRNA expression in the rat mammary gland at different activation stages

Leptin is expressed in various tissues, suggesting that this protein is effective not only at the central nervous system level, but also peripherically. Recent studies have shown leptin production by other tissues, including the placenta, stomach, and mammary tissues, but there is no information available concerning expression levels of leptin in the rat mammary gland at different activation stages. We used semi-quantitative RT-PCR to investigate leptin mRNA expression levels in the rat mammary gland at different activity stages. Rat mammary gland samples were collected from virgin females and on days 6, 12, 18 of pregnancy and of lactation (six rats per group). The expression levels of leptin mRNA were measured by semi-quantitative RT-PCR, with β-actin as an internal control. Leptin mRNA was highly expressed in virgin rat mammary glands (leptin(IOD)/β-actin(IOD) = 1.60). It decreased gradually during pregnancy, being lowest at 18 days of pregnancy, when the levels were significantly lower than in virgin mammary tissue. Leptin mRNA increased slightly during lactation, but the difference was not significant. By day 18 of lactation, expression levels of leptin mRNA reached the same values as in virgin mammary tissue (leptin(IOD)/β-actin(IOD) = 1.65). Based on these results, we suggest that leptin has an important regulation role in rat mammary gland activation.     

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, beta-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-x(L)were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R(3)-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues.     

Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor alpha and Epidermal Growth Factor Receptor mRNAs in Mice

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 mug / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor alpha (TGFalpha) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFalpha mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFalpha on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFalpha on the mammary gland growth. OVX resulted in a significant decrease of TGFalpha mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.     

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.     

Effect of chronic suppression of growth hormone secretion, by SMS 201-995 on lactation in mice

The effect of chronic suppression of growth hormone (GH) secretion by SMS 201-995 on lactation was studied in primiparous C3H/He mice. Mammary gland DNA content on day 12 of lactation was significantly lower in SMS 201-995 treated mice than in the control. There were little differences between groups in mammary gland RNA content and litter growth on day 12 of lactation. That was associated with a slightly higher RNA/DNA ratio and a significant increase in food intake during lactation. These results indicate that inhibited mammary gland growth by GH suppression has little effect on lactation. The smaller mammary gland can compensate by increasing its secretory activity.