Developmental regulation of calcium-binding proteins (calelectrins and calpactin I) in mammary glands

We recently showed that mammary glands contain a novel class of calcium-binding proteins (CBPs) that bind to membranes in a calcium-dependent manner. We have also established that these mammary CBPs are equivalent to the calelectrins and calpactin I/p36. Since it has been suggested that these proteins might be involved in exocytosis, we examined mammary glands for these CBPs during secretory differentiation. Immunohistochemical examination showed glands from virgin animals to be rich in calelectrins and calpactin I/p36, while glands from lactating animals contained little immunoreactive material. In addition, silver-staining and immunoblot estimation of the CBPs in lysates from collagenase harvested secretory epithelia showed these proteins to be significantly reduced compared to nonsecretory epithelia. Close examination of the CBP immunoreactive cells of the mammary gland shows that ductal cells are prominent in their staining and that the immunoreactive material is associated with the cell surface. Also, in juvenile glands the myoepithelial stem cells (cap cells) of the elongating end bud are devoid of the CBPs. In contrast to the in vivo data, epithelia cultivated on collagen gels demonstrate comparable levels of the CBPs in both nonsecretory and secretory monolayers. The in vivo data indicate that the CBPs are developmentally regulated during mammary gland differentiation such that secretory epithelia are essentially devoid of these novel proteins. Furthermore, a role for calelectrin and calpactin I/p36 in exocytotic casein secretion is questioned.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Abstract. Calelectrins are a family of antigenically related Ca²⁺-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca²⁺. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Expression of a putative stem cell marker,Musashi 1, in mammary glands of ewes

Several recent studies demonstrated that development, function and remodelling of mammary glands involved multipotent cells, but no specific molecular markers for mammary epithelial stem cells were revealed. These studies principally concerned human and mouse mammary tissue, but mammary stem cells could be a valuable tool in agricultural production and bioengineering in farm animals. The Musashi-1 (Msi 1) gene encodes an RNA binding protein, which is likely to be associated with self-renewal of neural, intestinal and mammary progenitor cells and is believed to influence the Notch signalling pathway. In this study Musashi-1 expression was detected using immunohistochemistry and in situ hybridisation analysis on mammary glands of ewes at different developmental stages. The protein expression was observed in the epithelial cells at all stages examined. In situ hybridization analysis showed that Msi 1 mRNA has an expression pattern similar to the encoded protein, with positive staining in both nuclei and cytoplasm of ductal, secretory and stromal cells. Ultrastructural in situ analysis confirmed the nuclear and cytoplasmatic expression of Msi. Quantitative analysis of Msi 1 gene expression showed a strong correlation with that of Ki-67, that is a marker of cell proliferation. This is the first report outlining expression of Msi 1 in ovine mammary glands during a complete cycle of lactation.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Calelectrins are a family of antigenically related Ca2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Mammary gland Ca2+-binding (-dependent) proteins: Identification as calelectrins and calpactin I/p36

Calcium-binding (-dependent) proteins (CBPs) associated with the spreading of mammary epithelial cell cultures have been identified as various calelectrins and calpactins (p36). In immunoblot analysis, the CBPs of 30–36 kD and 68–70 KD variously react with different calelectrin and calpactin I monomer/p36 antisera. The same immunoreactive proteins were shown to be present in virgin mammary glands and collagen gel mouse mammary epithelial cell cultures. The mammary CBPs show extensive immunochemical relatedness; however, they fail to show cross-reaction with antiserum to calpactin II (lipocortin) antiserum. These immunoreactive CBPs comigrate in electrophoresis with ³⁵S-methionine-labeled CBPs isolated from mammary epithelial cell cultures. Unlike calmodulin, the mammary CBPs that correspond to calelectrins and calpactin I monomer/p36 are not stable to thermal denaturation. The mammary CBPs bind to epithelial cell membranes in a Ca²⁺-dependent manner and are differentially released from ruptured cells, compared with calmodulin, suggesting subcellular localization. Phenothiazineagarose and phenylagarose are equivalent in their ability to bind the mammary CBPs. Thus, mammary gland CBPs of 30–36 kD and 68–70 kD have been shown to be related or equivalent to the calelectrins and to calpactin I monomer/p36. Since these proteins are known to bind Ca²⁺, we conclude that the mammary gland CBPs are also Ca²⁺-binding proteins. The mammary gland CBPs are immunologically related and probably represent members of a larger family of related proteins.     

Peroxisome proliferator-activated receptor-binding protein null mutation results in defective mammary gland development

A conditional null mutation of peroxisome proliferator-activated receptor-binding protein (PBP) gene was generated to understand its role in mammary gland development. PBP-deficient mammary glands exhibited retarded ductal elongation during puberty, and decreased alveolar density during pregnancy and lactation. PBP-deficient mammary glands could not produce milk to nurse pups during lactation. Both the mammary ductal elongation in response to estrogen treatment and the mammary lobuloalveolar proliferation stimulated by estrogen plus progesterone were attenuated in PBP-deficient mammary glands. The proliferation index was decreased in PBP-deficient mammary glands. PBP-deficient mammary epithelial cells expressed abundant beta-casein, whey acidic protein, and WDNM1 mRNA, indicating a relatively intact differentiated function. PBP-deficient epithelial cells were unable to form mammospheres, which were considered to be derived from mammary progenitor/stem cells. We conclude that PBP plays a pivotal role in the normal mammary gland development.     

67 k calcimedin (67 kDa) is distinct from p67 calelectrin and lymphocyte 68 kDa Ca2+-binding protein.

The 67 k calcimedin is a Ca2+-binding protein present in both muscle cells and peritoneal macrophages. Many tissues, including lymphoid tissues, liver and lymphocytes, have been shown to contain Ca2+-binding proteins of similar molecular size, such as the p67(67 kDa) calelectrin or the 68 kDa lymphocyte protein. We have tested affinity-purified antibodies raised to the smooth-muscle 67 k calcimedin in these several tissues and here report that the 67 k calcimedin is not detectable in liver, thymus, spleen or thymic lymphocytes. These findings support recent biochemical evidence, discussed here, suggesting that the 67 k calcimedin is a protein different from calelectrin and the 68 kDa lymphocyte protein. The more limited tissue distribution of the 67 k calcimedin, which includes muscle and macrophages, suggests that the 67 k calcimedin may function in Ca2+-mediated events special to these cell types. The affinity-purified antibodies to the 67 k calcimedin will be useful in obtaining information concerning the special roles of this Ca2+-binding protein in these cells.     

Isolation of two 67 kDa calcium-binding proteins from pig lung differing in affinity for phospholipids and in anti-phospholipase A2 activity

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium-dependent affinity chromatography on polyacrylamide-immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant-associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholipase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+-binding protein (67E), whose role still remains to be determined.     

Electron microscopic localization of calelectrin, a Mr 36 000 calcium-regulated protein, at the cholinergic electromotor synapse of Torpedo

Calelectrin is a calcium-binding protein of Mr 36 000 which has previously been shown to be associated with membranes of the cholinergic synapse in a calcium-dependent manner. We report here that calelectrin was solubilized from the electric organ of Torpedo marmorata in the absence of calcium together with proteins of Mr 54 000 and Mr 15 000. In cholinergic nerve endings isolated from the electric organ only calelectrin was solubilized in a calcium-dependent manner. A specific antiserum to calelectrin was used to localize the antigen by immunofluorescence microscopy on sections of electric organ and showed that calelectrin is distributed throughout the postsynaptic cell. Calelectrin was also detected in axons and in the cell bodies of the cholinergic neurones where it was concentrated in discrete patches throughout the cells. Electric organ tissue was processed to localize calelectrin with the electron microscope using an immunoperoxidase method. The most intense staining was observed on the cytoplasmic face of the acetylcholine receptor-containing postsynaptic membrane and also associated with the intracellular filaments of the electrocyte. The intensity of staining associated with these structures could be greatly reduced by preincubating the tissue with calcium chelators. In nerve terminals calelectrin was associated with synaptic vesicles in a polarized fashion. Calelectrin was also found on the cytoplasmic face of the synaptosomal plasma membrane and associated with neurofilaments. No extracellular staining was ever observed. Our results strongly support our original hypothesis that calelectrin is a calcium-regulated component of intracellular structure associated both with membranes and filaments.     

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.     

Disruption of reelin signaling alters mammary gland morphogenesis

Reelin signaling is required for appropriate cell migration and ductal patterning during mammary gland morphogenesis. Dab1, an intracellular adaptor protein activated in response to reelin signaling, is expressed in the developing mammary bud and in luminal epithelial cells in the adult gland. Reelin protein is expressed in a complementary pattern, first in the epithelium overlying the mammary bud during embryogenesis and then in the myoepithelium and periductal stroma in the adult. Deletion in mouse of either reelin or Dab1 induced alterations in the development of the ductal network, including significant retardation in ductal elongation, decreased terminal branching, and thickening and disorganization of the luminal wall. At later stages, some mutant glands overcame these early delays, but went on to exhibit enlarged and chaotic ductal morphologies and decreased terminal branching: these phenotypes are suggestive of a role for reelin in spatial patterning or structural organization of the mammary epithelium. Isolated mammary epithelial cells exhibited decreased migration in response to exogenous reelin in vitro, a response that required Dab1. These observations highlight a role for reelin signaling in the directed migration of mammary epithelial cells driving ductal elongation into the mammary fat pad and provide the first evidence that reelin signaling may be crucial for regulating the migration and organization of non-neural tissues.     

Stem cell characteristics of transplanted rat mammary clonogens

Rat mammary glands contain a subpopulation of clonogenic epithelial cells with large proliferation and differentiation potentials. When transplanted, the clonogens in monodispersed rat mammary epithelial cell suspensions give rise to either alveolar units (AUs) or ductal units (DUs) depending on the nature of the hormonal milieu in the graft recipient. Clonogens are also the primary cells of origin of mammary cancer following exposure to ionizing radiation or chemical carcinogens. Given the other stem cell characteristics of mammary clonogens, it would be expected that the primary AUs and DUs to which they give rise when grafted and hormonally stimulated (a) would be derived from the same clonogenic cell subpopulation, (b) would contain all of the functionally differentiated cell types of homologous parts of comparably stimulated mammary glands in situ, and (c) would also contain clonogen subpopulations capable when subtransplanted of giving rise to secondary AUs and DUs of similar cell composition. The current experiments were designed to test these expectations. The data are discussed in the context of results of previous studies with this and other experimental models. The results further support the conclusion that rat mammary clonogens are multipotent mammary stem cells.     

An immunological comparison of several novel calcium-binding proteins

Polyclonal antibodies prepared against each of the calcimedins were utilized to determine their tissue distribution. The immunological survey of rat tissues revealed that the levels of the 35-kDa calcimedin varied, while the amount of the 67-kDa calcimedin was relatively constant in the tissues examined. A new immunoreactive species, 52 kDa, was detected with the antibody to the 35-kDa calcimedin; this protein appears to be the predominant immunoreactive species in the tissues examined. Antibodies to the 35-kDa calcimedin were also used to compare many other calcium-binding proteins in order to determine immunological relationships. These comparisons demonstrate that the epidermal growth factor receptor/kinase substrate (p35), the src kinase substrate (pp36), and calregulin are immunologically unrelated to the calcimedins. However, it was found that the 67-kDa calcimedin and the p70 calelectrin are identical, as are the 35-kDa calcimedin and the p32.5 calelectrin. The calimedins are a subset of the chromobindins. In addition, the antibody to the 35-kDa calcimedin also cross-reacts with synexin, which may be related to the new 52-kDa immunoreactive protein identified.     

The use of thioesterase II as a rat mammary epithelial cell-specific marker

The avidin-biotin-peroxidase complex immunoperoxidase technique was employed to determine the intercellular distribution of thioesterase II in rat mammary glands. This enzyme is responsible for shifting the product specificity of the fatty-acid synthetase enzyme complex from long to medium chain fatty acids. Thioesterase II was found exclusively in the cells lining the lumen of the ductal and alveolar structures in glands from mature virgin (150 days old) and pregnant rats. The ductal cell staining intensity was considerably less than that of the alveolar cells in the mature virgin rat glands. No immunoreactive thioesterase II was found in the stromal, adipose, vascular, or myoepithelial components of the gland in the developmental stages examined. In the glands from immature virgin rats (40-45 days old) thioesterase II was again found only in the epithelial cells lining the lumen of the ductal and end-bud structures although this layer was usually more than one cell thick. Quantitative determination of thioesterase II activity in cytosol preparations revealed similar levels in mammary fragments from enzymatically-dissociated glands obtained from mature virgins and in end buds derived from immature virgins, but somewhat higher levels in mammary structures derived from late-pregnant animals. These immunohistological and biochemical results demonstrate thioesterase II's usefulness as a mammary epithelial cell-specific marker.     

Isolation and characterization of two novel calcium-dependent phospholipid-binding proteins from bovine lung

Two calcium-dependent proteins of apparent Mr 32,000 and 34,000 were isolated from bovine lung. Approx. 70 mg/kg of each was obtained. Two-dimensional gel electrophoresis in the presence of 8 M urea showed their apparent p/values to be 5.1 and 5.0, respectively. Both proteins are related immunologically to calelectrin from Torpedo marmorata. They also have very similar amino acid compositions to calelectrin. Partial sequence information shows that both proteins contain the highly conserved sequence described for the annexins, a new family of calcium-dependent membrane-binding proteins. In common with other members of this family, the new proteins bind to acidic phospholipids in a calcium-dependent manner.     

Murine Mammary Epithelial Stem Cells: Discovery,Function, and Current Status

An entire mammary epithelial outgrowth, capable of full secretory differentiation, may comprise the progeny of a single cellular antecedent, i.e., may be generated from a single mammary epithelial stem cell. Early studies showed that any portion of an intact murine mammary gland containing epithelium could recapitulate an entire mammary epithelial tree on transplantation into an epithelium-free mammary fat pad. More recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium and that their behavior and maintenance is dependent on signals generated both locally and systemically. In this review, we have attempted to develop the scientific saga surrounding the discovery and characterization of the murine mammary stem/progenitor cell hierarchy and to suggest further approaches that will enhance our knowledge and understanding of these cells and their role in both normal development and neoplasia.Before the 1980s there was little if any thought that the epithelium in murine mammary glands might be engendered by or supported by a mammary epithelial specific stem cell. In 1980, Rudland et al. wrote a review entitled “Stem cells in rat mammary development and cancer: A review” and noted that dimethylbenz [α] anthracene (DMBA)-induced rat carcinomas contained all three main types of epithelium found in the normal rat gland, those lining the ductal lumina, those lining the alveolar lumina, and myoepithelial cells (Rudland et al. 1980). They suggested, based on the two types of morphologically distinct epithelial (luminal and myoepithelial) cancer cells in the clonally derived Rama 25 cell line, that a single cell might give rise to both types and this also held true when these cells were inoculated into hosts and produced tumors. Williams and Daniel (Williams and Daniel 1983) suggested that the cap cells at the tip of the growing ducts in the mouse could give rise to both luminal and myoepithelial cells during ductal morphogenesis. However, no direct evidence that a single cell could produce both epithelial cell types in vivo was available. Nevertheless, in retrospect there was evidence that full regenerative activity for mammary epithelial existed in every part of the adult mammary epithelial tree.The experiments that originally showed the potential existence of stem cells in the mouse mammary gland were the pioneering studies of DeOme and his students, Les Faulkin and Charles Daniel. The approach they developed and optimized was serial transplantation of normal mammary gland into the cleared mammary fat pad of syngeneic mice (Deome et al. 1959; Faulkin and Deome 1960). The cleared mammary fat pad allowed the transplantation and growth of normal mammary cells into their normal anatomical site and under the influence of a normal physiological environment. Using this method, DeOme and coworkers showed that all portions of the normal mammary gland contains cells that will grow and fill the fat pad with a normal ductal mammary tree and respond to hormones with a normal differentiation program (Daniel 1975; Daniel et al. 1975). The progeny of the transplanted cells could be serially transplanted into the appropriate recipients for multiple times; however, unlike preneoplastic or neoplastic cells, the normal cells always senesced after multiple serial transplants, generally five to eight transplant generations (Daniel 1975). This was interpreted as indicating mammary stem cells possessed a finite proliferative activity (i.e., life span). This finite life span was a fundamental difference between normal and preneoplastic/neoplastic mammary cells. Cells with an indefinite in vivo life span (i.e., immortalized) have been identified in numerous mammary model systems, including MMTV-induced alveolar hyperplasia''s (Callahan and Smith 2000), chemical carcinogen-induced ductal and alveolar hyperplasia''s (Smith et al. 1978, 1980), hormonally induced alveolar hyperplasia, spontaneously immortalized ductal hyperplasia''s (Medina 2000, 2002), and cells containing specific genetic alterations (i.e., p53 deletion, Polyoma mT antigen) (Maglione et al. 2001; Medina et al. 2002).Subsequent studies showed that stem cells were located along the entire mammary tree and represented in all the different developmental states of the mammary gland. These stages included primary and tertiary ducts from 6- and 16-wk virgin glands, uniparous and multiparous regressed gland, 15-d pregnant and 10-d lactating glands (Smith and Medina 1988). Host age and reproductive history had little influence on the frequency of stem cells as measured by percent successful takes and life span assay (Young et al. 1971; Smith and Medina 1988). Mammary cells taken from 26-mo-old virgin mice had the same transplant potential as cells taken from 3-wk-old mice. Cell populations, from both, senesced after five transplant generations. Similarly, mammary cells in 12-mo-old multiparous mice had the same serial transplant potential as cells from 3-wk-old virgin mice (Young et al. 1971). Finally, continuous hormone stimulation did not induce additional loss of ductal growth potential. These results have important implications for understanding the role of mammary stem cells in normal mammary development because they emphasize that the mammary stem cell is a relatively quiescent cell that is only activated under conditions of gland repopulation (i.e., fetal growth stage and pubertal growth phase).     

Identification of Cell Types in the Developing Goat Mammary Gland

The goat was chosen as the model system for investigating mammary gland development in the ruminant. Histological and immunocytochemical staining of goat mammary tissue at key stages of development was performed to characterize the histogenesis of the ruminant mammary gland. The mammary gland of the virgin adult goat consisted of a ductal system terminating in lobules of ductules. Lobuloalveolar development of ductules occurred during pregnancy and lactation which was followed by the regression of secretory alveoli at involution. The ductal system was separated from the surrounding stroma by a basement membrane which was defined by antisera raised against laminin and Type IV collagen. Vimentin, smooth-muscle actin and myosin monoclonal antisera as well as antisera to cytokeratin 18 and multiple cytokeratins stained a layer of myoepithelial cells which surround the ductal epithelium. Staining of luminal epithelial cells by monoclonal antibodies to cytokeratins was dependent on their location along the ductal system, from intense staining in ducts to variable staining in ductules. The staining of epithelial cells by monoclonals to cytokeratins also varied according to the developmental status of the goat, being maximal in virgin and involuting glands, lowest at lactation and intermediate during gestation. In addition, cuboidal cells, situated perpendicular to myoepithelial cells and adjacent to alveolar cells in secretory alveoli, were also stained by cytokeratin monoclonal antibodies and antisera to the receptor protein, erbB-2, in similar fashion to luminal epithelial cells. These results demonstrate that caprine mammary epithelial cell differentiation along the alveolar pathway is associated with the loss of certain types of cytokeratins and that undifferentiated and secretory alveolar epithelial cells are present within lactating goat mammary alveoli.     

Null mutation of peroxisome proliferator-activated receptor-interacting protein in mammary glands causes defective mammopoiesis

To investigate the role of nuclear receptor coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) in mammary gland development, we generated a conditional null mutation of PRIP in mammary glands. In PRIP-deficient mammary glands, the elongation of ducts during puberty was not affected, but the numbers of ductal branches were decreased, a condition that persisted long after puberty, indicating that the potential of ductal branching was impaired. During pregnancy, PRIP-deficient mammary glands exhibited decreased alveolar density. The lactating PRIP-deficient glands contained scant lobuloalveoli with many adipocytes, whereas the wild type glands were composed of virtually no adipocytes but mostly lobuloalveoli. As a result, PRIP mammary-deficient glands could not produce enough milk to nurse all the pups during lactation. The ductal branching of mammary glands in response to estrogen treatment was attenuated in PRIP mutant glands. Whereas the proliferation index was similar between wild type and PRIP-deficient glands, increased apoptosis was observed in PRIP-deficient glands. PRIP-deficient glands expressed increased amphiregulin, transforming growth factor-alpha, and betacellulin mRNA as compared with wild type glands. The differentiated function of PRIP-deficient mammary epithelial cells was largely intact, as evidenced by the expression of abundant beta-casein, whey acidic protein (WAP), and WDNM1 mRNA. We conclude that PRIP is important for normal mammary gland development.     

Mouse mammary epithelial cells express the Na-K-Cl cotransporter,NKCC1: characterization,localization, and involvement in ductal development and morphogenesis

Despite the fact that physiological evidence points to the existence of a functional Na-K-Cl cotransporter in the mammary gland, the molecular identity of this transport process remains unknown. We now show that the Na-K-Cl cotransporter isoform, NKCC1, is expressed in mammary tissue. Developmental profiling revealed that the level of NKCC1 protein was significantly influenced by the stage of mammary gland development, and immunolocalization studies demonstrated that NKCC1 was present on the basolateral membrane of mammary epithelial cells. To examine whether functional NKCC1 is required for mammary epithelial cell development, we used NKCC1 -/- mice. We demonstrate that NKCC1 -/- mammary epithelium exhibited a significant delay in ductal outgrowth and an increase in branching morphogenesis during virgin development. These effects were autonomous to the epithelium as assessed by mammary gland transplantation. Although the absence of NKCC1 had no apparent effect on gross mammary epithelial cell morphology during lactation, pups born to NKCC1 -/- mice failed to thrive. Finally, analysis of NKCC1 protein in mouse models that exhibit defects in mammary gland development demonstrate that high levels of NKCC1 protein are indicative of ductal epithelial cells, and the presence of NKCC1 protein is characteristic of mammary epithelial cell identity.