Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.     

Precocious Mammary Gland Development in P-Cadherin–deficient Mice

To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin– deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin–null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin–null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin–mediated adhesion and/or signals derived from cell–cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.     

Expression of P-cadherin distinct from that of E-cadherin in re-epithelialization in neonatal rat skin

Our previous study showed that an open wound made in neonatal rat skin was covered by migration of certain undifferentiated populations of keratinocytes as a multilayered cell sheet. In this study, the expression of the components of adherens junctions (AJ), E- and P-cadherins, and beta-catenin, was examined to understand the underlying mechanisms. Both E- and P-cadherins were downregulated in the basal layer at 6 h post-wounding (PW), indicating a reduction in the intercellular adhesiveness. The expression of P-cadherin but not E-cadherin was expanded to the suprabasal layers at the wound margin at 12 h PW. Moreover, the expression pattern of P-cadherin at sites of cell-cell contact was punctate rather than linear. By 24 h PW, cells accumulated beta-catenin in the cytoplasm in a suprabasal layer contacting the basal layer at the wound margin. Both the E- and P-cadherins showed a punctate AJ pattern at the confined suprabasal layer. Such differential expression of the E- and P-cadherins strongly suggests that these two classic cadherins play distinct roles in re-epithelialization. The changing of the E- and/or P-cadherin expression may participate in a delay of terminal differentiation of keratinocytes for cell supply toward a wound.     

P-cadherin controls the differentiation of oral keratinocytes by regulating cytokeratin 1/10 expression via C/EBP-beta-mediated signaling

P-cadherin belongs to the family of Ca²⁺-dependent homophilic glycosylated cell adhesion molecules. In the normal oral epithelium it shows a strong expression in the basal cell layer which gradually decreases in the suprabasal cell layers. The exact role of P-cadherin during the development and homeostasis of the oral epithelium has not been elucidated, yet. Here, we show for the first time that P-cadherin controls differentiation by regulating cytokeratin (CK) 1/10 expression in primary oral keratinocytes (POK) from normal, but interestingly not in POKs from oral squamous cell carcinoma (OSCC) tissue. SiRNA knockdown of P-cadherin in normal POKs revealed a strong upregulation of CK1/10 expression on mRNA and protein level. In contrast, E-cadherin knockdown in normal oral keratinocytes did not show any influence on CK1/10 expression. Moreover, in comparison with normal control keratinocytes normal oral keratinocytes with reduced P-cadherin expression displayed an enhanced expression and a stronger nuclear staining of C/EBP-beta, a well-known regulator of CK1/10 expression in keratinocytes. Furthermore, after P-cadherin knockdown in normal POKs the promoter activity of a C/EBP-responsive luciferase construct was significantly higher than in normal POKs with regular P-cadherin expression. Additionally, we noticed a proliferation advantage in normal oral keratinocytes in contrast to keratinocytes with diminished P-cadherin expression. However, the inverted effect was seen in tumor derived primary oral keratinocytes. In summary, we show that P-cadherin contributes to the keratinocyte differentiation in the oral epithelium by influencing the CK1 and CK10 expression via C/EBP-beta-mediated signaling in normal but not in tumor derived oral keratinocytes from OSCC patients.     

Organotypic growth and differentiation of human mammary gland in sponge-gel matrix supported histoculture

Summary Blocks of breast tissue obtained during radical mastectomies from 23 patients with mammary gland carcinomas were used for cultivation in native-state, gel-supported histocultures. We show that the human mammary gland can be successfully maintained in this system so that normal epithelial breast structures proliferate and undergo differentiation for several weeks and a well-developed system of ducts and lobules is formed. Using antibodies to individual keratins 17 and 8 we have shown for the first time that ducts and alveoles developing in vitro undergo differentiation into the lining epithelium and myoepithelium in the same way as mammary gland epithelium in vivo. Growth of epithelial structures in vitro is also accompanied by the development of continuous basal membrane.     

The pattern of beta-catenin responsiveness within the mammary gland is regulated by progesterone receptor

Experiments involving beta-catenin loss- and gain-of-function in the mammary gland have decisively demonstrated the role of this protein in normal alveologenesis. However, the relationship between hormonal and beta-catenin signaling has not been investigated. In this study, we demonstrate that activated beta-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of DeltaN89beta-catenin. Cells at ductal tips are inherently beta-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers beta-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by beta-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling.     

Squamous cell carcinoma and mammary abscess formation through squamous metaplasia in Smad4/Dpc4 conditional knockout mice

Smad4 is a central mediator for TGFbeta signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, we disrupted this gene in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. We demonstrated that absence of Smad4 resulted in beta-catenin accumulation at onset and throughout the process of transdifferentiation, implicating beta-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. We further demonstrated that TGFbeta1 treatment degraded beta-catenin and induced epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions were blocked in the absence of Smad4. These findings indicate that TGFbeta/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia.     

乳腺生物反应器特异高效表达载体的构建

乳腺生物反应器具有广阔的开发前景,而提高目的基因的表达量是该领域一个重要的研究课题。因此,选用强的非特异性启动子pCAG,而不是乳腺特异性启动子来实现高效表达;通过Cre/loxP系统和山羊β-乳球蛋白启动区（pBLG）表达Cre重组酶来实现载体的自删除以达到乳腺特异表达的目的。方法：构建含PolyA终止信号的Cre乳腺特异表达元件PolyA-pBLG-cre,插至强启动子pCAG驱动的报告基因lacZ表达载体中,构建成乳腺特异表达载体pCBCZ（pCAG-loxP-PolyA-pBLG-cre-loxP-lacZ）。转染细胞实验结果：PCR鉴定确认pCBCZ载体在小鼠乳腺上皮细胞（HC11）中发生Cre-loxP同源重组。X-Gal染色表明载体能驱动lacZ在HC11细胞中高效表达β-半乳糖苷酶,而在NIH 3T3细胞中仅少量表达。结论：构建的pCBCZ载体能高效驱动外源基因在乳腺细胞中表达,且具有较好的乳腺特异性,为研发乳腺生物反应器表达载体提供新的方法。     

Soluble fragment of P-cadherin adhesion protein found in human milk

Classical cadherins such as E- and P-cadherin are transmembrane proteins that mediate specific cell-to-cell adhesion and are important to tissue development and function. Cadherin function can be modulated by various means, including proteolytic cleavage of the extracellular adhesion domain from the cells' surface, yielding large soluble fragments termed (soluble) sE- or sP-cadherin. In people with certain carcinomas, sE-cadherin can be detected at elevated levels in the serum and sometimes can serve as a prognostic marker. Soluble E-cadherin also is found in urine of patients with bladder cancer. In addition to being present in bodily fluids of cancer patients, sE- and sP-cadherin are present in the serum of healthy people, suggesting that shedding of cadherins is a normal event. Here, we report high levels of 80 kDa sP-cadherin in human milk. In the lactating mammary gland tissue, P-cadherin appears to be a protein secreted by epithelial cells, rather than an adhesion protein. This is in contrast to the non-lactating mammary gland where P-cadherin is restricted to myoepithelial cells, and is present at sites of cell-cell contact.     

Cadherins and the mammary gland

Cadherin cell-cell adhesion proteins are critical for the formation of tissues from single cells. E-and P-cadherin play important roles in the architecture and function of the normal mammary gland. In breast cancers, the expression, or lack thereof, of E-cadherin can differentiate tumor types, whereas the misexpression of either P-cadherin or N-cadherin can be a marker of poor prognosis or increased malignancy, respectively. Additional research is needed to more precisely define the roles of both classical and desmosomal cadherins and their downstream signaling events, in the development and malignant behavior of breast cancers.     

Functional analysis of mouse keratin 8 in polyoma middle T-induced mammary gland tumours

Keratin 8 and 18 are commonly used as tumorigenic markers for various types of carcinomas. They are known to be involved in cell migration, cell invasiveness, plasminogen activity and drug and radiation resistance. To ascertain a potential function for simple epithelium keratins in mammary adenocarcinoma in vivo, keratin-8-deficient mice (mK8) were mated with transgenic mice carrying the middle T oncogene driven by the MMTV promoter. The resulting mK8 knockout and control progeny carrying the middle T transgene developed mammary gland tumours with the same incidence. However, the onset of palpable mammary gland tumours occurred earlier in mK8 mutant than in control mice. This effect was prominent in males where the onset in control animals is delayed overall, because of the lower hormonal inducibility of the MMTV promoter. Metastatic foci were observed in the lungs of all females and of a few males, idependently of the genotype. Histological analysis revealed no morphological differences of the tumorigenic cells in primary tumours nor in metastatic foci. As expected, keratin 8 was absent in the mK8 tumours. Keratin 7 (mK7), keratin 18 (mK18) and keratin 19 (mK19) protein were observed in both primary and metastatic foci. These results constitute the first in vivo analysis of the role of simple epithelium keratins in mammary carcinogenesis. It demonstrates that the latency, but not the incidence nor the morphological features, of PyV middle T-induced mammary gland tumours is affected by keratin 8 deficiency     

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.