Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca²⁺ concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.     

Identification and partial characterization of mesenchyme-derived growth factor that stimulates proliferation and inhibits functional differentiation of mouse mammary epithelium in culture

The effect of mesenchyme on both proliferation and differentiation of mammary epithelial cells was investigated in a primary cell culture system. Mammary cells cultured on collagen gel for 4 days produced casein in response to the synergistic action of insulin, cortisol, and prolactin. When mammary epithelial cells were co-cultured with fibroblasts derived from three different kinds of fetal mesenchymal tissues, casein production was suppressed. The addition of conditioned media obtained from cultures of these mesenchymal cells stimulated DNA synthesis and reduced casein synthesis in a dose-dependent fashion in the cultured mammary cells. Although such biological actions are similar to those of epidermal growth factor (EGF), the capability to compete with EGF for EGF receptor was not found in this conditioned medium. Sephadex G-200 column chromatography revealed that molecular weight of the peak which has these biological activities was around 100,000. These results indicate that fetal mesenchymal cells secrete a substance(s) which has a stimulatory effect on proliferation and an inhibitory effect on differentiation of mammary epithelial cells.     

Adipocyte-epithelial interactions regulate the in vitro development of normal mammary epithelial cells

Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complement in vivo reconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.     

Adipocyte–Epithelial Interactions Regulate thein VitroDevelopment of Normal Mammary Epithelial Cells

Mammary epithelial organoids (MEO), isolated from pubescent rats, were cultured within a reconstituted basement membrane in transwell inserts, in the presence or absence of mature mammary adipocytes in the lower well. This system allowed for free medium exchange between the two compartments, without direct cell-to-cell contact. When cultured in serum-free medium supplemented with insulin, prolactin, hydrocortisone, progesterone, and various epidermal growth factor (EGF) concentrations, mammary adipocytes did not affect epithelial cell growth, but enhanced epithelial differentiation. Casein and lipid accumulations were monitored as indicators of functional differentiation of MEO. Mammary adipocytes significantly enhanced casein and lipid accumulation within the MEO, independently of EGF concentration. Furthermore, adipocytes induced MEO to preferentially undergo alveolar morphogenesis, inhibited squamous outgrowth, and increased lumen size. These findings demonstrate that morphological and functional differentiation of mammary epithelial cells is profoundly enhanced by the adipose stroma and that these effects are mediated by diffusible paracrine factors. This new model can be exploited in future studies to define the mechanisms whereby hormones and growth factors regulate mammary gland development and carcinogenesis. Moreover, it could complementin vivoreconstitution/transplantation studies, which are currently employed to evaluate the role of specific gene deletions in the regulation of mammary development.     

Is EGF a physiological inhibitor of mouse mammary casein synthesis? Unphysiological responses to pharmacological levels of hormones

It has been observed that EGF inhibits the induction of casein synthesis by mouse mammary tissue in vitro in addition to acting as a promoter of mammary epithelial proliferation. However, since the circulating level of EGF increases during lactation, and since functional EGF receptors are retained by the lactating cells, it seemed unlikely that EGF is an inhibitor of mammary differentiation in vivo. The current studies demonstrate, in fact, that EGF inhibits the induction of casein synthesis in vitro only when insulin is present in the culture medium at unphysiologically high concentrations. Other artifactual responses to high levels of hormones are described.     

Growth of epithelium from a preneoplastic mammary outgrowth in response to mammary adipose tissue

Summary We investigated the effects of conditioned media derived from mouse mammary fat pads on the proliferation of CL-S1 cells, an epithelial cell line originally isolated from a preneoplastic mammary outgrowth line. Cell proliferation in vitro in serum-free defined medium was compared to that in this medium conditioned using intact mammary fat pad pieces or isolated fat pad adipocytes. Culture medium was conditioned by incubating the conditioning material in defined culture medium for 24 h at 37°C. Conditioned medium induced CL-S1 proliferation as much as 10- to 20-fold above the minimal levels of growth in control cultures after 13 d of culture. The growth-stimulatory factor(s) had an apparent molecular weight of greater than 10 kDa. This growth-stimulatory activity was both heat and trypsin stable. Because the role of adipose tissue is to store and release lipids, we next tested whether lipids are released during medium conditioning. The lipid composition of the fat pad conditioned medium was characterized using both thin layer and gas liquid chromatography. These lipid analyses indicated that the fat pad pieces released significant amounts of fatty acids and phospholipids into the medium during the conditioning period. The free fatty acid composition included both saturated and unsaturated molecules, and about 80% of the total fatty acids consisted of palmitate, stearate, oleate, and linoleate. These same fatty acids were a structural component of the majority of phospholipid found in the medium. The addition of palmitate or stearate to defined medium had no effect or was inhibitory for CL-S1 proliferation, depending on the concentration used. Defined medium supplemented with oleate, arachidonate, or linoleate induced CL-S1 proliferation, and the inhibitory effects of palmitate and stearate were overcome by addition of oleate and linoleate. These data indicate that both unsaturated and saturated fatty acids are released from intact adipose cells of the mouse mammary fat pad and that fatty acids can influence the growth of prenoplastic mouse mammary epithelium. Thus, unsaturated fatty acids, perhaps in conjunction with other substances released simultaneously, are candidate molecules for the substances that mediate the effect of adipose tissue on growth of epithelium. This work was supported in part by a grant from the American Institute for Cancer Research; grant CA 46885 from the National Institutes of Health, Bethesda, MD; and by State of Washington initiative 171.     

Further characterization of the inhibition of casein production in a primary mouse mammary epithelial cell culture by epidermal growth factor. Antagonism by cyclic AMP

Epidermal growth factor (EGF) inhibited casein production and the accumulation of casein mRNA activity induced by insulin (I), cortisol (F) and prolactin (P) in a primary culture of mammary epithelial cells from pregnant mice. The inhibitory effects of EGF were blocked by 8-bromo cyclic AMP (8-br-cAMP) in a dose-dependent manner. The effect of 8-br-cAMP was observed at a concentration as low as 20 microM and was maximal at 500 microM. Dibutyryl cyclic AMP (db-cAMP), cAMP, and 3-isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, also antagonized the inhibitory effect of EGF on casein production. 8-Br-cAMP had, however, no effect on the mitogenic activity of EGF in this system. These results suggest a possible modulatory role of cAMP in EGF-induced inhibition of casein production in cultured mammary epithelial cells.     

Mammary organoids from immature virgin rats undergo ductal and alveolar morphogenesis when grown within a reconstituted basement membrane

We have recently described a primary culture system which allows for extensive proliferation and functional differentiation of immature mammary epithelial cells. Herein, these findings are extended to demonstrate that a distinct pattern of ductal and alveolar morphogenesis can be induced within the mammary organoids isolated from virgin female rats and cultured within an Engelbreth-Holm-Swarm sarcoma-derived reconstituted basement membrane under defined serum-free conditions. The lobular and multilobular organoids that emerged resemble the alveoli of the mammary gland in gross form, multicellular architecture, and cytologic and functional differentiation, while the ductal organoids expressed characteristics typical of mammary gland ducts in vivo. The epithelial cells within the alveolar- and duct-like organoids displayed the capability of secreting two morphologically distinct milk products, casein and lipid, into the luminal compartment. The expression of histiotypic morphogenesis and mammary-specific functional differentiation by the cultured mammary organoids proceeded in the absence of a morphologically distinct basal lamina. We illustrate that development highly reminiscent of that which naturally occurs in the mammary gland in vivo can be induced and supported in vitro under defined serum-free conditions. In addition, the methodologies are available to simultaneously monitor mammary organoid morphogenesis, growth, and functional differentiation. This system should serve as a unique model in which the regulation of branching morphogenesis, development, gene expression, and transformation can be examined.     

Prolactin and epidermal growth factor regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01–10 μg/ml) and EGF (1–100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland. © 1995 Wiley-Liss, Inc.     

Prolactin sensitivity of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol

We examined the responsiveness to prolactin and growth hormone of mammary epithelial cells from mice exposed neonatally to diethylstilbestrol (DES) and from control mice. The mammary epithelial cells were cultured inside collagen gels with serum-free medium containing insulin, epidermal growth factor, and linoleic acid. This produces prolactin-sensitive cells with low levels of casein production, as measured in cellular homogenates with a specific enzyme-linked immunosorbent assay for alpha-casein. The collagen gels containing these cells were then released and the medium supplements changed to insulin, linoleic acid, and prolactin at concentrations from 10 to 1000 ng/ml and growth hormone at 0, 10, or 100 ng/ml. This second phase of the culture, the differentiation phase, allows the cells to accumulate casein if they have this capacity. When cultured with prolactin only (no growth hormone), the cells from DES-exposed mice consistently accumulated 50-100% of the casein content of normal cells, but never more. Growth hormone, when added to prolactin-containing medium, increased casein accumulation above the levels seen with prolactin alone. Combinations of prolactin and growth hormone enhanced the difference between casein accumulation in DES-exposed and control cells, and DES-exposed cells were much less responsive to growth hormone. In our studies, the isolated mammary epithelial cells of estrogen-exposed mice are not more sensitive to prolactin than cells from normal animals as previous reports reports had suggested, but rather are generally less sensitive to hormonal stimulants.     

Measurement by radioimmunoassay of casein content in rabbit mammary gland during pregnancy and after prolactin stimulation in organ culture

A specific homologous radioimmunoassay was developed to measure rabbit beta-casein in rabbit mammary gland with a sensitivity of 0.5 ng/ml protein. It was used to measure casein concentration during pregnancy and in organ culture of mammary gland explants. Casein was detectable in virgin mammary glands, showed a small increase during the first half of pregnancy, increased more than 20-fold between Days 21 and 27, and diminished somewhat on the first days of lactation. After 24 hr of culture, mammary gland explants had no detectable casein, but the addition of increasing concentrations of prolactin to a culture medium which contained insulin (5 micrograms/ml) and cortisol (0.5 microgram/ml) induced a regular increase in the casein content of the tissue. Casein started to increase when 10 ng/ml of prolactin was present and maximal values were achieved for 100 ng/ml of the hormone.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Effect of tubulozole, a new synthetic microtubule inhibitor, on the induction of casein gene expression by prolactin

Colchicine and related drugs are known to inhibit milk secretion. They are also able to prevent stimulation of casein and DNA synthesis by prolactin in the mammary gland. The present report reports data obtained with tubulozole, a new antimitotic drug. Tubulozole C added to culture medium of isolated rabbit epithelial mammary cells strongly inhibited their multiplication. Simultaneously, at a concentration of 1 microM, it prevented almost completely the induction of beta-casein mRNA. Induced cells were rapidly deinduced by addition of the drug to the medium. A similar inhibition was observed when the induction was obtained with prolactin alone or with its two stimulators insulin and glucocorticoids. Tubulozole T, an isomer of tubulozole C which is known to be ineffective in disrupting microtubules, did not alter prolactin actions. These data and those obtained with other tubulin-binding drugs strongly suggest that the integrity of microtubules is required for prolactin to deliver its message to the mammary cell.     

Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy

Epidermal growth factor stimulated cell proliferation in a primary mammary epithelial cell culture derived from mice at different stages of pregnancy. Moreover, the peptide hormone inhibited casein production induced by the synergistic actions of insulin, cortisol and prolactin. The inhibitory effect of epidermal growth factor was influenced by the gestational stages of the mammary gland. These effects of epidermal growth factor were exerted at physiological concentrations. The dual actions of epidermal growth factor on mammary cells implicate its participation in regulation of the growth and differentiation of the mammary gland during pregnancy.     

Regulation of hormone-sensitive lipase expression by saturated fatty acids and hormones in bovine mammary epithelial cells

Hormone-sensitive lipase was firstly identified as an epinephrine-induced lipase in adipocyte. HSL mRNA was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and bovine lactating mammary gland. Saturated fatty acids (stearate and palmitate), but not unsaturated fatty acids (oleate and linoleate) induced up-regulation of HSL mRNA in a time- and concentration-dependent manner in bMEC. Treatment with insulin (5-10 ng/ml), dexamethasone (50-250 nM) or GH (50 ng/ml) induced down-regulation of HSL. These results suggest that HSL was regulated by fatty acids and some hormones in mammary epithelial cells and thereby play an important role of lipid and energy metabolism.     

INHIBITION OF LACTOGENIC ACTIVITIES OF BOVINE MAMMARY GLAND EXPLANTS BY THE WHEY FRACTION OF BOVINE MILK

The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. α-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on α-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the β-lactoglobulin gene was found.     

Regulation of glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1) gene in the mouse mammary gland differs from that of casein genes

Mouse glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), also known as mC26 and homologous to bovine PP3, is a milk protein synthesized in the mammary gland. Several studies have investigated the regulation of casein, the major milk protein, gene in the mammary gland, but little is known about GlyCAM-1. Here we examined GlyCAM-1 gene expression in mouse mammary epithelial cells. First, we detected GlyCAM-1 expression in mammary epithelial cells in situ by immunohistochemistry; almost all mammary epithelial cells of the lactating mouse expressed GlyCAM-1. Second, mammary epithelial cells were digested with collagenase and cultured with insulin, prolactin and/or glucocorticoid. alpha-Casein and beta-casein genes were expressed following treatment with insulin, prolactin and glucocorticoid. In contrast, GlyCAM-1 expression could not be detected with any combination of these three hormones. We also analyzed changes in the levels of GlyCAM-1 and caseins mRNAs in cultured cells. The addition of hormones to the culture medium increased casein mRNAs, but surprisingly reduced GlyCAM-1 mRNA. Our results suggest that the mechanisms that regulate GlyCAM-1 gene in mammary cells of lactating mice are different from those involved in the regulation of casein genes.     

Effect of ouabain on the lactogenic action of prolactin and on the level of mammary prolactin receptors

Ouabain added to the culture medium of rabbit mammary gland inhibits prolactin action on the initiation of lactose and casein synthesis. The degree of inhibition is a function of the ouabain concentration in the medium. Likewise, ouabain blocks the accumulation of casein mRNA supported by prolactin. In addition, ouabain provokes a rapid disappearance of prolactin receptors. Conversely prolactin keeps its capacity to enhance the concentration of casein mRNA and the parallel casein synthesis when K+ ions are totally absent from the culture medium. These results suggest that although prolactin induces a modification of the K+/Na+ ratio in the mammary cell and ouabain prevents this effect of prolactin, the inhibitory action of ouabain on lactogenesis can be explained essentially by its effect on the hormone receptors.