乳腺脂肪组织调节乳腺发育的研究进展

乳腺是哺乳动物哺育子代的重要器官,其通过分泌乳汁给子代提供充足的营养物质,乳腺的健康发育对泌乳以及提高子代的存活率具有重要意义.脂肪组织是乳腺重要的组成部分,在乳腺发育和循环重构过程中,乳腺脂肪组织随之呈现规律性的形态和功能变化,乳腺脂肪组织的动态变化是乳腺循环性发育重构的重要特征.脂肪组织能够分泌特殊的"脂肪因子"调节上皮细胞的功能和乳腺的发育,并且存在与上皮细胞相互转换的潜能.本综述综合近年来乳腺脂肪组织的相关研究进展,为后续研究脂肪组织调节乳腺发育的机制提供基础数据.     

Cleavage of Histone 3 by Cathepsin D in the Involuting Mammary Gland

The post-lactational regression of mammary gland is a complex multi-step process designed to conserve the biological function of the gland for next pregnancy. This developmental stage is a biological intrigue with great relevance to breast cancer research, and thus has been the subject of intensive scrutiny. Multipronged studies (microarray, proteomics profiling, animal knock-out models) have provided a repertoire of genes critical to involution. However, the caveat of these approaches remains in their failure to reveal post-translational modification(s), an emerging and critical aspect of gene regulation in developmental processes and mammary gland remodeling. The massive surge in the lysosomal enzymes concurrent with the onset of involution has been known for decades, and considered essential for “clearance” purposes. However, functional significance of these enzymes in diverse biological processes distinct from their proteolytic activity is just emerging. Studies from our laboratory had indicated specific post-translational modifications of the aspartyl endopeptidase Cathepsin D (CatD) at distinct stages mammary gland development. This study addresses the biological significance of these modifications in the involution process, and reveals that post-translational modifications drive CatD into the nucleus to cleave Histone 3. The cleavage of Histone 3 has been associated with cellular differentiation and could be critical instigator of involution process. From functional perspective, deregulated expression and increased secretion of CatD are associated with aggressive and metastatic phenotype of breast cancer. Thus unraveling CatD’s physiological functions in mammary gland development will bridge the present gap in understanding its pro-tumorigenic/metastatic functions, and assist in the generation of tailored therapeutic approaches.     

Protein kinase expression during murine mammary development

The susceptibility of the mammary gland to carcinogenesis is influenced by its normal development, particularly during developmental stages such as puberty and pregnancy that are characterized by marked changes in proliferation and differentiation. Protein kinases are important regulators of proliferation and differentiation, as well as of neoplastic transformation, in a wide array of tissues, including the breast. Using a RT-PCR-based cloning strategy, we have identified 41 protein kinases that are expressed in breast cancer cell lines and in the murine mammary gland during development. The expression of each of these kinases was analyzed throughout postnatal mammary gland development as well as in a panel of mammary epithelial cell lines derived from distinct transgenic models of breast cancer. Although the majority of protein kinases isolated in this screen have no currently recognized role in mammary development, most kinases examined were found to exhibit developmental regulation. After kinases were clustered on the basis of similarities in their temporal expression profiles during mammary development, multiple distinct patterns of expression were observed. Analysis of these patterns revealed an ordered set of expression profiles in which successive waves of kinase expression occur during development. Interestingly, several protein kinases whose expression has previously been reported to be restricted to tissues other than the mammary gland were isolated in this screen and found to be expressed in the mammary gland. In aggregate, these findings suggest that the array of kinases participating in the regulation of normal mammary development is considerably broader than currently appreciated.     

mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.

BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.     

Role of ovarian secretions in mammary gland development and function in ruminants

The mammary gland is a dynamic organ that undergoes cyclic developmental and regressive changes during the lifetime of a female mammal. Mammogenesis begins during embryonic life with the development of the first mammary gland rudiments and ductal system. After birth, during the pre-pubertal period, the ductal growth of the mammary parenchyma occurs through the fat pad. In most of the ruminant species allometric mammary parenchyma development begins with the onset of cyclic ovarian secretions activity. The two main hormones secreted during an ovarian cycle are estradiol and progesterone. These steroid hormones are derived from cholesterol and are synthesized by theca and granulosa cells in ovaries. During puberty, the mammary parenchyma develops in a compact, highly arborescent parenchymal mass surrounded by a dense connective matrix. Ductal elongation and lobulo-alveolar development are accomplished during growth and pregnancy to prepare for future milk production. At the end of lactation, the mammary gland undergoes involution, which corresponds to a regression of the secretory tissue, a reduction in the alveolar size and a loss of mammary epithelial cells (MECs). Ovarian steroids (estradiol and progesterone) appear to be key regulators of the different stages of mammogenesis and mammary function. Through this review, the role and the importance of ovarian steroids on mammary gland and on MECs is described.     

HIF1alpha is a critical regulator of secretory differentiation and activation,but not vascular expansion,in the mouse mammary gland

During pregnancy the mammary epithelium and its supporting vasculature rapidly expand to prepare for lactation, resulting in dramatic changes in the micro-environment. In order to investigate the role of oxygenation and metabolism in these processes, the oxygen-responsive component of the hypoxia-inducible factor (HIF) 1 complex, HIF1alpha, was deleted in the murine mammary gland. Although vascular density was unchanged in the HIF1alpha null mammary gland, loss of HIF alpha impaired mammary differentiation and lipid secretion, culminating in lactation failure and striking changes in milk composition. Transplantation experiments confirmed that these developmental defects were mammary epithelial cell autonomous. These data make clear that HIF1alpha plays a critical role in the differentiation and function of the mammary epithelium.     

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.     

Developmental stage determines the effects of MYC in the mammary epithelium

Epidemiological findings suggest that the consequences of a given oncogenic stimulus vary depending upon the developmental state of the target tissue at the time of exposure. This is particularly evident in the mammary gland, where both age at exposure to a carcinogenic stimulus and the timing of a first full-term pregnancy can markedly alter the risk of developing breast cancer. Analogous to this, the biological consequences of activating oncogenes, such as MYC, can be influenced by cellular context both in terms of cell lineage and cellular environment. In light of this, we hypothesized that the consequences of aberrant MYC activation in the mammary gland might be determined by the developmental state of the gland at the time of MYC exposure. To test this hypothesis directly, we have used a doxycycline-inducible transgenic mouse model to overexpress MYC during different stages of mammary gland development. Using this model, we find that the ability of MYC to inhibit postpartum lactation is due entirely to its activation within a specific 72-hour window during mid-pregnancy; by contrast, MYC activation either prior to or following this 72-hour window has little or no effect on postpartum lactation. Surprisingly, we find that MYC does not block postpartum lactation by inhibiting mammary epithelial differentiation, but rather by promoting differentiation and precocious lactation during pregnancy, which in turn leads to premature involution of the gland. We further show that this developmental stage-specific ability of MYC to promote mammary epithelial differentiation is tightly linked to its ability to downregulate caveolin 1 and activate Stat5 in a developmental stage-specific manner. Our findings provide unique in vivo molecular evidence for developmental stage-specific effects of oncogene activation, as well as the first evidence linking MYC with activation of the Jak2-Stat5 signaling pathway.     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

Development and loss of androgen responsiveness in the embryonic rudiment of the mouse mammary gland

The androgen-responsive phase in the development of the mammary gland was determined by exposing rudiments of various developmental stages to testosterone in vitro. Although testosterone causes destruction of the mammary epithelium in 14-day male fetuses, it failed to prevent formation of mammary buds in explanted 11-day skin. It was found that mammary rudiments become responsive to androgens only late in Day 13 of gestation, and that they are no longer responsive on Day 15 and later. Both acquisition and loss of androgen responsiveness do occur on time in explanted glands, indicating intrinsic developmental changes in the rudiment. The experiments and their results are schematically summarized in Fig. 2.     

Connexins: a junctional crossroad to breast cancer

The mammary gland presents a valuable model for developmental studies, spanning the embryonic stage through menarche to menopause. The dynamic remodeling of this gland is orchestrated by cellular heterogeneity, integrating mammogenic, systemic and local cues. Gap junctional intercellular communication provides pivotal cross talk of mammary epithelial cells with the surrounding cells and their local microenvironment. Connexins are involved in regulating normal and pathological mammary gland development, through channel-dependent and channel-independent roles. Modulation of the isoforms of connexins expressed, as well as their differential assembly into connexons and recruitment of a variety of associated partners, contributes to the complexity of signaling relayed at the membrane. This confers context-dependent functions of connexins at different stages of development and carcinogenesis. This review will summarize available knowledge about the functional dynamics of connexins and gap junctions in regulating normal mammary gland development and its pathophysiology.     

Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.     

Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture.

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradation of the ECM during the different stages of mammary development. Mammary tissue extracts from virgin and pregnant CD-1 mice resolved by zymography contained three major proteinases of 60K (K = 10(3) Mr), 68K and 70K that degraded denatured collagen. These three gelatinases were completely inhibited by the tissue inhibitor of metalloproteinases. Proteolytic activity was lowest during lactation especially for the 60K gelatinase which was shown to be the activated form of the 68K gelatinase. The activated 60K form decreased prior to parturition but increased markedly after the first two days of involution. An additional gelatin-degrading proteinase of 130K was expressed during the first three days of involution and differed from the other gelatinases by its lack of inhibition by the tissue inhibitor of metalloproteinases. The activity of the casein-degrading proteinases was lowest during lactation. Three caseinolytic activities were detected in mammary tissue extracts. A novel 26K cell-associated caseinase--a serine arginine-esterase--was modulated at different stages of mammary development. The other caseinases, at 92K and a larger than 100K, were not developmentally regulated. To find out which cell type produced the proteinases in the mammary gland, we isolated and cultured mouse mammary epithelial cells. Cells cultured on different substrata produced the full spectrum of gelatinases and caseinases seen in the whole gland thus implicating the epithelial cells as a major source of these enzymes. Analysis of proteinases secreted by cells grown on a reconstituted basement membrane showed that gelatinases were secreted preferentially in the direction of the basement membrane. The temporal pattern of expression of these proteinases and the basal secretion of gelatinases by epithelial cells suggest their involvement in the remodelling of the extracellular matrix during the different stages of mammary development and thus modulation of mammary cell function.     

The extracellular matrix as an adhesion checkpoint for mammary epithelial function

The development of the mammary gland is spatially regulated by the interaction of the mammary epithelium with the extracellular matrix (ECM). Cells receive cues from the ECM through a family of adhesion receptors called integrins, consisting of alpha- and beta-chain dimers. Integrins assist cells in sensing their appropriate developmental context in response to both hormones and growth factors. Here we argue that cell adhesion to the ECM plays a key role in specific developmental checkpoints, particularly in alveolar survival, morphogenesis and function. Specific ablation of alphabeta1-integrins in the luminal epithelium of the mammary gland shows that this sub-type of receptors is required for proliferation, accurate morphological organisation, as well as milk secretion. Downstream, small Rho GTPases mediate cellular polarisation and differentiation. Current challenges in studying the integration of signals in checkpoints of mammary gland development are discussed.     

Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis to carcinogenesis

The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme.