Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model

Development of the mammary gland during puberty, pregnancy, and lactation is controlled by steroid and peptide hormones and growth factors. To determine the role of the insulin-like growth factors (IGFs) in this process we developed a transgenic model using the whey acidic protein (WAP) gene to direct expression of rat IGF-I and human IGF binding protein-3 (IGFBP-3) to mammary tissue during late pregnancy and throughout lactation. High levels of expression of transgenic IGF-I and IGFBP-3 were seen in lobular-alveolar cells by in situ hybridization. There was no obvious effect on mammary development during pregnancy and lactation; indeed, mothers were capable of nursing their pups normally and the only structural difference seen in the mammary glands at peak lactation was an overall smaller size of the alveoli. We also evaluated the role of IGF-I and IGFBP-3 in the remodeling of mammary tissue during involution. Compared with control animals, the process of involution was modified in both transgenic lines. The degree of apoptotic cells was lower in the WAP-IGF-I and WAP-BP-3 expressing mice. In addition, there was a more quiescent pattern of involution with residual lobular secretary ability and a muted host inflammatory reaction with fewer lumenal microcalcifications. These results demonstrate that IGF-I and IGFBP-3 may modulate the involutionary process of the lactating mammary gland.     

Effect of stages of lactation on the concentration of a 90-kilodalton heat shock protein in bovine mammary tissue

From the normal mammary tissue of a Holstein cow in late lactation, a heat shock protein (90 kDa) was purified by ammonium sulfate fractionation and five-step column chromatography. From 70 g of tissue, 9.5 mg of this heat shock protein were obtained; samples had 98% purity and 19% recovery. The molecular mass of the 90-kDa heat shock protein was estimated to be 86 kDa by SDS-PAGE. Analysis of the amino-terminal amino acid sequence suggested that the protein had been purified as a mixture of two isoforms. The contents of the heat shock protein in cytoplasmic fractions of mammary tissues from Holstein heifers and cows at lactation and involution were measured by quantitative immunoblot analysis using rabbit antiserum raised against the purified heat shock proteins. The contents of the heat shock protein were higher in tissues from lactating cows than in those from heifers and involuting cows. The elevated concentrations of cytoplasmic 90-kDa heat shock protein in lactating tissue suggested that this protein is involved in mammary differentiation and lactation.     

Delayed involution of the mammary epithelium in BALB/c-p53null mice

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been defined. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21WAF1 mRNA. Induction of p21WAF1 mRNA was absent in BALB/c-p53null mice, and therefore, was shown to be p53-dependent. The BALB/c-p53null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53(wt) mice through 5 days post-weaning. The delay was transient with no differences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaffected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the first stage of involution initiated by the epithelium itself, but does not affect the second phase during which stromal proteases are induced.     

Active and tissue inhibitor of matrix metalloproteinase-free gelatinase B accumulates within human microvascular endothelial vesicles

Human gelatinase B is involved in tissue remodeling and angiogenesis. It is thought to be synthesized and rapidly secreted as an inactive precursor. In this report, we have shown that human endothelial cells accumulate active forms of gelatinase B in the cytosol. Microvascular but not macrovascular endothelial cells dramatically increased the expression of cytosolic gelatinase B in response to phorbol myristate acetate. Western blotting showed that tissue inhibitor of metalloproteinase-1 (TIMP1) was also present in the cytosol. Whereas gelatinase B was complexed with TIMP1 in the conditioned medium, it existed as a free enzyme in the cytosol, suggesting that the formation of gelatinase B and TIMP1 complex occurs after their secretion. Immunogold electron microscopy revealed that gelatinase B was localized in secretory vesicles which were especially prominent in invading pseudopodia. In contrast, TIMP1 was found throughout the cytoplasm but was not present in the gelatinase vesicles. The accumulation of intracellular activated gelatinase B, ready for rapid release, may facilitate the migration of microvascular endothelial cells during angiogenesis.     

Identification of lactoferrin complexes in bovine mammary secretions during mammary gland involution

Part of the antimicrobial activity of lactoferrin resides in its ability to bind to bacteria. The complexing of lactoferrin with other proteins could alter its activity. This study identified the presence of lactoferrin complexes in mammary secretions during mammary gland involution and determined the proportion of free and complexed lactoferrin in mammary secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution. Proteins were fractionated from defatted, filtered mammary secretions by sucrose density gradient ultracentrifugation and by gel filtration chromatography. Proteins contained in separated fractions were identified by SDS-PAGE. The presence of lactoferrin was confirmed by immunoblot analysis. Lactoferrin was present as complexed forms of high molecular mass in mammary secretions at each day of involution. The majority of lactoferrin was present in complexes of higher molecular mass rather than as monomers. A majority of lactoferrin existed in fractions of approximately 250 kDa, although peaks of lactoferrin at 150, 300, and 800 kDa were also found. The presence of lactoferrin complexes may result from interactions with casein or immunoglobulins or from the formation of lactoferrin multimers in the secretions. The interaction of lactoferrin with other proteins in mammary secretions during involution may affect the antimicrobial properties of lactoferrin.     

Osteopontin expression in mammary gland development and tumorigenesis

Osteopontin (OPN) is a secreted phosphoprotein that binds to cells via an Arg-Gly-Asp sequence and to mineralized surfaces. The protein can mediate cell adhesion and is strongly implicated in transformation and tumorigenesis. We have examined the expression pattern of OPN in mouse mammary glands at different stages of postnatal development. Whereas OPN is expressed at low-to-moderate levels in mammary glands from virgin and pregnant mice, the levels of OPN mRNA are extremely high in the lactating gland, consistent with the presence of the protein in milk. Expression is highest at 2 days of lactation and declines thereafter, but it remains high through involution. OPN expression is restricted to small nests or groups of cells at 9 days of involution. These results suggest that OPN may play a specific role in the process of involution that may be distinct from its role during lactation. In mammary tumors arising spontaneously in transgenic mice expressing the oncogenes c-myc and/or v-Ha-ras under the control of the mouse mammary tumor virus promoter, the level of OPN expression is increased dramatically over that in the normal gland in these same animals. Numerous cells expressing OPN mRNA are widespread throughout the tumors. OPN protein is detectable by Western blotting in extracts from the mammary gland at 2 days of lactation and from the tumors, but not in mammary glands at other stages of development. We hypothesize that OPN is exported from most tissues and that the protein is only detectable in tissues elaborating fluids, such as the lactating mammary gland, or in pathological situations when expression of OPN is abnormally high, such as in tumors.     

In situ localization of WDNM1 and ferritin heavy chain gene expression in mammary gland

In situ hybridization was performed with sections obtained from mammary gland at 10 days of lactation and at 1 and 3 days of involution using either digoxigenin-labeled antisense or sense RNA probe in order to localize expression of WDNM1 and ferritin heavy chain mRNA. The WDNM1 gene was predominantly expressed in the layer of secretory epithelial cells surrounding the lumen of mammary gland alveoli. The lower levels of WDNM1 mRNA were observed at involution day 3 compared to involution day 1. The expression of ferritin heavy chain mRNA also appears to be confined to the epithelial layer of mammary alveoli. The lower levels of ferritin heavy chain mRNA were observed at involution day 3 compared to involution day 1.     

An in vitro system for the study of matrix metalloproteases during decidualization in the mouse

Decidualization results in the remodeling of the extracellular matrix with the loss of collagen type I and the appearance of basement membrane matrix components. We have developed an in vitro assay system to study matrix metalloproteases during mouse decidualization. Uterine stroma, or decidua isolated from day 7.5 pregnant mice, were grown on a three-dimensional collagen type I matrix (Vitrogen). Gelatin zymography of conditioned media from these cultures showed constitutive secretion of processed forms of gelatinase A at 65, 62, and 59 kDa with 62 kDa predominating. Similar patterns of gelatinase A expression were obtained from tissue lysates of decidualizing uteri from days 5.5 to 7.5 of development. Cells cultured on Vitrogen, but not on plastic or matrix-coated dishes, were able to process the proenzyme to the 59 kDa form as observed in vivo. Only stroma cells cultured on a coating of collagen type I displayed the same increase in the 59 kDa zymogen. Decidua cells grown on Vitrogen attached and then migrated into aggregates that eventually penetrated the gel and spread as differentiated decidua on the underlying plastic. These preliminary results suggested that the in vitro assay system can be used to study the role of metalloproteases in matrix remodeling during decidualization.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

p96, a MAPK-related protein, is consistently downregulated during mouse mammary carcinogenesis

Differential display PCR [DD-PCR] was applied to identify mRNAs differentially expressed between two consecutive stages of an in vivo model of mouse mammary carcinogenesis. The extended life 12 [EL12] and transformed mammary 12 [TM12] outgrowths differ in morphology, ovarian hormone dependence, and tumorigenicity, yet the TM12 outgrowth arose spontaneously from the EL12 outgrowth. A fragment of the mouse p96 gene was identified using DD-PCR. The differential expression of p96 was confirmed using RNase protection assays. Examination of the RNA expression patterns of the p96 isoforms during normal mammary gland development showed high levels in the involuting mammary gland and in preneoplastic hyperplasias. In contrast, p96 isoform mRNA levels were consistently decreased in mammary tumors derived from the in vivo hyperplasias. Examination of p96 protein levels revealed a decrease in p96 protein in a number of mammary tumors as compared to their hyperplastic precursors further supporting the observations that p96 gene expression is consistently downregulated in mammary tumors. The functional activity of p96 protein has not been resolved, however the observation that p96 gene expression is downregulated in two different tumor systems (human ovarian tumors and mouse mammary tumors) warrants more extensive investigation on its role in normal and neoplastic cell growth.     

Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland

The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C-terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.     

Distribution and origin of peptide-containing nerve fibres in the rat and human mammary gland

The structures in the mammary gland involved in milk ejection have been investigated with regard to their relation to different types of peptidergic nerve fibres and their origin. Lactating rats were studied with immunohistochemistry focusing on the nipple, the parenchyma, the mammary blood vessels and the mammary nerve. The human mammary gland was also analysed. In the mammary gland from rat and human, nerve endings in the subepidermis, around smooth muscle cells in the nipple, in the connective tissue surrounding lactiferous ducts and alveoli in the nipple and in the parenchyma of the mammary gland showed immunoreactivity for calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, peptide histidine isoleucine, neuropeptide Y, galanin and tyrosine hydroxylase, whereas dynorphin-positive nerve fibres could not be detected. The mammary nerve contained calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y and tyrosine hydroxylase immunoreactivities; the adventitia of the mammary artery contained nerve fibres immunoreactive for neuropeptide Y and tyrosine hydroxylase, while vasoactive intestinal polypeptide-, peptide histidine isoleucine-, calcitonin gene-related peptide- and substance P-positive fibres were found in the tissue surrounding the artery. The wall of the mammary vein had nerve terminals immunoreactive for neuropeptide Y, tyrosine hydroxylase, calcitonin gene-related peptide and substance P. With the help of retrograde tracing using wheat germ agglutinin in combination with immunohistochemistry, projections of calcitonin gene-related peptide-immunoreactive cells in the dorsal root ganglia to the nipple were established. Neurons in the sympathetic stellate ganglion containing neuropeptide Y and tyrosine hydroxylase also projected to the mammary gland. Moreover retrogradely-labelled cells were found in the nodose ganglion, and they were vasoactive intestinal polypeptide-immunoreactive. These results demonstrate a rich distribution of different types of nerve fibres in structures of the mammary gland related to milk ejection. These nerve fibres and their peptides may be involved in the local control of milk ejection.     

Sialomucin complex, a heterodimeric glycoprotein complex. Expression as a soluble, secretable form in lactating mammary gland and colon

Ascites 13762 rat mammary adenocarcinoma cells express abundantly on their cell surfaces a heterodimeric glycoprotein complex composed of a sialomucin ascites sialoglycoprotein (ASGP)-1 and a transmembrane subunit ASGP-2. The latter, which contains two epidermal growth factor-like domains, binds the receptor tyrosine kinase p185(neu), suggesting that the complex is bifunctional as well as heterodimeric. Immunoblot analyses using monoclonal antibodies prepared against the complex demonstrate high levels of expression in rat lactating mammary gland and colon. Immunolocalization studies with anti-ASGP-2 indicate that ASGP-2 is present in these two tissues in the apical regions of secretory epithelial cells. Both mammary gland and colon contain a soluble, secretable form of ASGP-2, which is not found in the ascites cells; milk and mammary gland also have the membrane form. Immunoblot analyses using a COOH-terminal-specific polyclonal antibody indicate that the soluble form of ASGP-2 is missing its COOH-terminal domains. Both the soluble and membrane forms of ASGP-2 are similar to the membrane-associated form from the 13762 adenocarcinoma with respect to Mr, antigenicity, and association with ASGP-1. The presence of ASGP-1 in milk suggests that it is a candidate for the uncharacterized high Mr milk mucin, MUCX. ASGP-2 expression is up-regulated in mammary gland during pregnancy, because it is undetectable in virgin and early pregnant rats but abundant in the gland from late pregnant and lactating animals. However, compared with the lactating mammary gland, the 13762 ascites cells overexpress ASGP-2 by more than 100-fold, which may contribute to their malignancy. These combined results indicate that sialomucin complex is a unique secreted product in the mammary gland and colon, whose behavior is different from that in the mammary ascites tumors, and which may play important roles in mammary and intestinal physiology.     

Regulation of branched-chain amino acid metabolism in the lactating rat

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1. 9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD.     

Basis for detection of stenosis using venous administration of microbubbles during myocardial contrast echocardiography: bolus or continuous infusion?

Recent studies have shown that the lactating mammary gland is able to utilize plasma-derived dipeptides for milk protein synthesis. However, it was not clear whether the peptides were hydrolysed followed by uptake of the constituent amino acids or were taken up intact. In view of this, we have designed experiments to investigate (a) whether the lactating rat mammary gland is capable of transporting hydrolysis-resistant dipeptides and (b) whether or not mammary cells are able to hydrolyse peptides, including glutathione, extracellularly. The uptake of the hydrolysis-resistant dipeptides D-[3H]Phe-L-Gln and D-[3H]Phe-L-Glu by the perfused rat mammary gland was low. Concomitant addition of L-Leu-L-Ala (50 mM) had no effect on the clearance of either labelled dipeptide suggesting that the small, albeit significant, uptake of the dipeptides is not via a high affinity peptide transporter (PepT1/PepT2). All anionic dipeptides tested (L-Glu-L-Ala, L-Asp-L-Ala, L-Ala-L-Asp, L-Asp-Gly, Gly-L-Asp and Gly-L-Glu) with the exception of D-Phe-L-Glu were able to trans-accelerate the efflux of labelled D-aspartate from preloaded rat mammary tissue (explants and perfused mammary gland). It appears that these peptides were being hydrolysed extracellularly followed by the uptake of free anionic amino acids via the mammary tissue high affinity, Na+-dependent anionic amino acid carrier operating in the exchange mode. Glutathione was able to trans-accelerate D-aspartate efflux from lactating rat mammary tissue in a fashion which was sensitive to the peptidase inhibitor acivicin. This suggests that gamma-glutamyltranspeptidase hydrolyses glutathione to produce L-glutamate which is subsequently transported via the high-affinity anionic amino acid carrier. Hydrolysis of peptides followed by uptake of the constituent amino acids may provide an important source of amino acids for milk protein synthesis.     

Functional receptors for atrial natriuretic peptide in the rat mammary gland during lactation

The present study was undertaken: 1) to localize and characterize atrial natriuretic peptide (ANP) receptors in the rat mammary gland; and 2) to elucidate ANP-induced cellular formation of cyclic GMP (cGMP) and alterations in alveolar morphology during both early and late lactation. Receptor autoradiography, employing rat-specific [125I]ANP as radioligand, demonstrated binding sites in the secretory tissue and larger blood vessels of the mammary gland. Binding of [125I]rANP to membrane fractions was completely displaced by unlabeled ANP and brain natriuretic peptide. C-type natriuretic peptide and cANP(4-23) revealed limited competition with radiolabeled ANP only during early lactation, indicating a more heterogeneous receptor population at that time. Systemically administered ANP induced cGMP formation in the alveolar epithelium, as shown with immunohistochemistry, and increased mammary tissue cGMP concentrations in vivo throughout the lactation period. Image analysis revealed enlargement of alveolar (but not epithelial) cell area after ANP stimulation in late lactation, suggesting altered alveolar filling or myoepithelial cell relaxation. These results indicate that ANP induces biological effects in the rat mammary gland through specific ANP-A receptor interaction with subsequent intracellular cGMP formation. ANP may therefore play a regulatory role in the control of mammary gland blood supply and secretory function.