Lactoferrin at basal side of mouse mammary epithelium derives in part from stroma cells

Lactoferrin is synthesized by glandular epithelial cells and neutrophils and is also present on both sides of the mammary epithelium. We have studied the origin of lactoferrin detected in the various compartments of mouse mammary tissue. As revealed by immunogold electron microscopy, lactoferrin is present in mammary epithelial cells and in the basal region of the epithelium, associated with connective tissue and stroma cells at all physiological stages studied. A perturbation of protein synthesis or transport after in vitro treatment with cycloheximide or brefeldin A does not abrogate lactoferrin labelling in the basal region of the epithelium. The expression of lactoferrin has also been observed in the fat pads of mammary glands from mice surgically depleted of epithelial cells. The sealing of one teat for 24 h is accompanied by an increase in both the number of stroma cells and the labelling of myoepithelial cells. Thus, the lactoferrin present in the interstitial space of the mouse mammary epithelium originates in part from stroma cells. Possible roles of lactoferrin at the basal side of the mammary epithelium are discussed.     

Bmi1 regulates stem cells and proliferation and differentiation of committed cells in mammary epithelium

PolycombGroup (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer [1]. Mice without the PcG gene Bmi1 are runted and suffer from progressive loss of hematopoietic and neural stem cells [2-4]. Here, we assess the effects of Bmi1 on stem cells and differentiation of an epithelial tissue in vivo. We chose the mammary gland because it allows limiting dilution transplantations [5, 6] and because Bmi1 is overexpressed in breast cancer [7, 8]. Our analyses show that Bmi1 is expressed in all cells of the mouse mammary gland and is especially high in luminal cells. Loss of Bmi1 results in a severe mammary-epithelium growth defect, which can be rescued by codeletion of the Ink4a/Arf locus or pregnancy. Even though mammary stem cells are present in the absence of Bmi1, their activity is reduced, and this is only partially due to Ink4a/Arf expression. Interestingly, loss of Bmi1 causes premature lobuloalveolar differentiation, whereas overexpression of Bmi1 inhibits lobuloalveolar differentiation induced by pregnancy hormones. Because Bmi1 affects not only mammary stem cells but also more committed cells, our data warrant a more detailed analysis of the different roles of Bmi1 in breast-cancer etiology.     

Mammospheres from murine mammary stem cell-enriched basal cells: Clonal characteristics and repopulating potential

Identification of murine mammary stem cells (MaSCs) has been attempted with various in vitro and in vivo assays. While, the in vivo repopulation assay remains as the most definitive assay for MaSC detection, it is expensive, time-consuming, and technically challenging. The in vitro mammosphere assay was considered unreliable because of major concerns about its clonal origin. In the current study, co-culture experiments with mammary cells from fluorescent protein transgenic mice and time-lapse video microscopy revealed that > 90% mammospheres formed from sorted basal epithelial-enriched cells were of clonal origin in terms of stem cell. These basal-cell derived mammospheres were further distinguished morphologically in a 3-dimensional extracellular matrix culture and functionally in the in vivo repopulation assay. Transplant of single mammospheres or the resultant 3-dimensional solid structures into gland-free mammary fat pads yielded a 70% success rate of multilineage mammary gland reconstitution. Thus, this in vitro sphere formation and differentiation assay is a reliable alternative to the in vivo repopulation assay for the study of MaSCs.     

Ablation of beta1 integrin in mammary epithelium reveals a key role for integrin in glandular morphogenesis and differentiation

Integrin-mediated adhesion regulates the development and function of a range of tissues; however, little is known about its role in glandular epithelium. To assess the contribution of beta1 integrin, we conditionally deleted its gene in luminal epithelia during different stages of mouse mammary gland development and in cultured primary mammary epithelia. Loss of beta1 integrin in vivo resulted in impaired alveologenesis and lactation. Cultured beta1 integrin-null cells displayed abnormal focal adhesion function and signal transduction and could not form or maintain polarized acini. In vivo, epithelial cells became detached from the extracellular matrix but remained associated with each other and did not undergo overt apoptosis. beta1 integrin-null mammary epithelial cells did not differentiate in response to prolactin stimulation because of defective Stat5 activation. In mice where beta1 integrin was deleted after the initiation of differentiation, fewer defects in alveolar morphology occurred, yet major deficiencies were also observed in milk protein and milk fat production and Stat5 activation, indicating a permissive role for beta1 integrins in prolactin signaling. This study demonstrates that beta1 integrin is critical for the alveolar morphogenesis of a glandular epithelium and for maintenance of its differentiated function. Moreover, it provides genetic evidence for the cooperation between integrin and cytokine signaling pathways.     

Beta1 integrin mediates internalization of mammalian reovirus

Reovirus infection is initiated by interactions between the attachment protein sigma1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein lambda2, which serves as the structural base for sigma1, suggests that integrins mediate reovirus endocytosis. A beta1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a beta1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in beta1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing beta1. However, reovirus bound equivalently to cells that differed in levels of beta1 expression, suggesting that beta1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into beta1-deficient cells was substantially diminished in comparison to viral uptake into beta1-expressing cells. These data provide evidence that beta1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.     

Characterization of stem cells from the murine adult mammary gland

The postnatal mammary morphogenesis comprises two steps, first, formation of a system of branching ducts at puberty and second, alveologenesis during pregnancy. The mammary epithelium is organized as a bilayer, composed of two cellular types, basal myoepithelial and luminal epithelial. The remarkable regenerative properties revealed in serial transplantation experiments suggest that the adult mammary epithelium harbors stem cells. Various strategies including analysis of DNA label-retaining cells, transgenic approach, and in vivo transplantation assay, have been used to isolate and characterize murine mammary stem and progenitor cells. Their molecular characteristics remain to be defined precisely but notable progress have been already made in the enrichment and identification of these cells. Current studies favor the hypothesis of a basal-type mammary stem cells expressing high levels of alpha 6, beta1 and beta 3 integrin chains, the major receptors of extracellular matrix proteins. Luminal-type progenitors may participate in the establishment of the bilayered alveolar epithelium during pregnancy.     

Identification and characterization of cancer initiating cells from BRCA1 related mammary tumors using markers for normal mammary stem cells

It is hypothesized that cancer stem cells arise either from normal stem cells or from progenitor cells that have gained the ability to self-renew. Here we determine whether mammary cancer stem cells can be isolated by using antibodies that have been used for the isolation of normal mammary stem cells. We show that BRCA1 mutant cancer cell lines contained a subpopulation of CD24+CD29+ or CD24+CD49f+ cells that exhibited increased proliferation and colony forming ability in vitro, and enhanced tumor-forming ability in vivo. The purified CD24+CD29+ cells could differentiate and reconstitute the heterogeneity found in parental cells when plated as a monolayer. Under low-attachment conditions, we detected “tumorspheres” only in the presence of double positive cells, which maintained their ability to self-renew. Furthermore, CD24+CD29+ cells could form tubular structures reminiscent of the mammary ductal tree when grown in three-dimensional cultures, implying that these cancer cells maintain some of the characteristics of the normal stem cells. Nevertheless, they could still drive tumor formation since as low as 500 double positive cells immediately after sorting from BRCA1 mutant primary tumors were able to form tumors with the same heterogeneity found in the original tumors. These data provide evidence that breast cancer stem cells originate from normal stem cells and advance our understanding of BRCA1-associated tumorigenesis with possible implications for future cancer treatment.     

Ultrastructure of the putative stem cell niche in rat mammary epithelium

There is now strong evidence that the stem cells of many tissues reside in specialized structures termed niches. The stem cell niche functions to house and regulate symmetric and asymmetric mitosis of stem cells in mammalian skin, mouse and human bone marrow, mouse brain, gut, and hair follicle, and Drosophila ovary and testis. This regulation is effected through the action of various signaling pathways such as Notch, Hedgehog, Wnt and others. The hormones of the estrous cycle, pregnancy and lactation that initiate growth in mouse mammary epithelium appear to act at a paracrine level to regulate mitosis through Notch receptors. Previous work has established that the putative stem cells of the mammary epithelium in several animal species reside near the basement membrane and never make contact with the ductal lumen. We show that these putative stem cells are found in anatomically specialized places created by the cytoplasmic extensions and modifications of neighboring differentiated cells. Such specializations may help to regulate stem cell activity by modulating molecular traffic to putative stem cells and contact with signaling molecules in the basement membrane. The histological characteristics of these putative niches vary as to the kinds of relationships the cells can have with the basement membrane and neighboring cells and as to how many stem or progenitor cells they may contain. This suggests a plasticity that may be relevant to the response of niches to tissue demands, such as wound healing, the periodic growth and regression of mammary epithelium, the process of mammary tumorigenesis therapeutic strategies for breast cancer.     

Beta1 integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli

Integrin-extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of beta1 integrin in the mammary gland, Itgbeta1(flox/flox) mice were crossed with WAPiCre transgenic mice, which led to specific ablation of beta1 integrin in luminal alveolar epithelial cells. In the beta1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell-basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in beta1 integrin mutant glands, which correlated with a specific increase of p21 Cip1 expression. In a p21 Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21 Cip1 to the proliferative defect observed in beta1 integrin mutant glands. A connection between p21 Cip1 and beta1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of beta1 integrin signaling in mammary epithelial cell proliferation.     

Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.     

Calpain restrains the stem cells compartment in breast cancer

CAPNS1 is essential for the stability and function of ubiquitous CAPN1 and CAPN2. Calpain modulates by proteolytic cleavage many cellular substrates and its activity is often deregulated in cancer cells, therefore calpain inhibition has been proposed as a therapeutical strategy for a number of malignancies. Here we show that CAPNS1 depletion is coupled to impairment of MCF7 and MCF10AT cell lines growth on plate and defective architecture of mammary acini derived from MCF10A cells. In soft agar CAPNS1 depletion leads to cell growth increase in MCF7, and decrease in MCF10AT cells. In both MCF7 and MCF10AT, CAPNS1 depletion leads to the enlargement of the stem cell compartment, as demonstrated by mammosphere formation assays and evaluation of stem cell markers by means of FACS and western blot analysis. Accordingly, activation of calpain by thapsigargin treatment leads to a decrease in the stem cell reservoir. The expansion of the cancer stem cell population in CAPNS1 depleted cells is coupled to a defective shift from symmetric to asymmetric division during mammosphere growth coupled to a decrease in NUMB protein level.     

Beta1 integrins regulate chondrogenesis and rock signaling in adipose stem cells

β1 integrins play a controversial role during chondrogenesis. Since the maturation of chondrocytes relies on a signaling switch from cell-cell to cell-matrix interactions, we hypothesized that β1 integrins play a different role at the earlier (mainly cell-cell interaction) from the later stage (mainly cell-matrix interaction) of chondrogenesis. Our data showed: in plain medium, sox9, collagen X, and collagen II gene expressions of ASCs were induced by β1-integrin blockage at day 14. In chondrogenic medium, however, sox 9, sox6, and collagen II gene expression were induced at day 4 but inhibited at day 14. In addition, both β1-integrin blockage and TGF-β1 down-regulated Rock-1 and -2 gene expression and produced the round cells. We concluded that β1 integrins play a more important role at the later stages than earlier stages of chondrogenesis, and that the onset of chondrogenesis promoted by β1-integrin blockage might be through inhibiting Rock signaling.     

The syndecan-1 ectodomain regulates alphavbeta3 integrin activity in human mammary carcinoma cells

The α_vβ₃ integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the α_vβ₃ integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to α_vβ₃ requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances α_vβ₃ recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt α_vβ₃-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated α_vβ₃ integrins.     

Functional and molecular characterization of rod-like cells from retinal stem cells derived from the adult ciliary epithelium

In vitro generation of photoreceptors from stem cells is of great interest for the development of regenerative medicine approaches for patients affected by retinal degeneration and for high throughput drug screens for these diseases. In this study, we show unprecedented high percentages of rod-fated cells from retinal stem cells of the adult ciliary epithelium. Molecular characterization of rod-like cells demonstrates that they lose ciliary epithelial characteristics but acquire photoreceptor features. Rod maturation was evaluated at two levels: gene expression and electrophysiological functionality. Here we present a strong correlation between phototransduction protein expression and functionality of the cells in vitro. We demonstrate that in vitro generated rod-like cells express cGMP-gated channels that are gated by endogenous cGMP. We also identified voltage-gated channels necessary for rod maturation and viability. This level of analysis for the first time provides evidence that adult retinal stem cells can generate highly homogeneous rod-fated cells.     

The basal lamina of the postnatal mammary epithelium contains glycosaminoglycans in a precise ultrastructural organization

The mammary epithelium was investigated to determine whether glycosaminoglycans (GAG) are components of the basal lamina of epithelia undergoing postnatal morphogenesis. Isolated epithelial tissues from midpregnant mice produce substantial amounts of GAG, consisting predominantly of hyaluronic acid and heparan sulfate. The basal surfaces of mammary epithelia at various postnatal developmental stages show GAG, as demonstrated by histochemistry and by autoradiography coupled with enzyme susceptibility. Electron microscopy using ruthenium red staining reveals polyanionic components, presumably GAG, within the epithelial basal lamina. Detailed ultrastructural analyses of tannic acid-treated and ruthenium red-stained material demonstrate that the lamina contains a two-dimensional symmetrical array of tetragonally ordered components colsely associated with the basal plasma membrane. This array is similar to that found in the hyaluronate-containing lamina of embryonic epithelia. A structurally ordered complex of GAG-containing macromolecules may characterize the basal lamina of all epithelia which undergo morphogenetic changes in cell shape.     

Beta1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and beta1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of beta1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in beta1 integrin-null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in beta1 integrin-null nerves improves sorting. Thus, increased activation of Rac1 by beta1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.     

Isolation of mouse mammary epithelial progenitor cells with basal characteristics from the Comma-Dbeta cell line

A mouse mammary epithelial cell line with morphogenetic properties in vivo, Comma-Dbeta, was used to isolate and to characterize mammary progenitor cells. We found that a homogeneous cell population expressing high surface levels of stem cell antigen 1 (Sca-1) was able to give rise in vivo to ductal and alveolar structures comprising luminal secretory and basal myoepithelial cells. Unlike the Sca-1(high), the Sca-1(neg/low) cell population displayed a reduced morphogenetic potential. The Sca-1(high) cells presented moderate CD24, high CD44 and alpha6 integrin surface levels, expressed basal cell markers p63, keratins 5 and 14, but no luminal and myoepithelial lineage markers. In culture, the Sca-1(high) cells generated identical daughter cells that retained their in vivo developmental potential, indicating that these cells were maintained by self-renewal. Plated at clonogenic density in Matrigel, Sca-1(high) cells formed spheroids that included luminal and myoepithelial cells. Thus, the isolated Sca-1(high) basal cells possess several features of stem/progenitor cells, including specific markers, self-renewal capacity, and the ability to generate the two major mammary lineages, luminal and myoepithelial. These data provide evidence for the existence of basal-type mouse mammary progenitors able to participate in the morphogenetic processes characteristic of mammary gland development.