Stimulation of dome formation in MDCK kidney epithelial cultures by inducers of differentiation: Dissociation from effects on transepithelial resistance and cyclic AMP levels

Changes in transepithelial electrical resistance and cyclic nucleotide levels were monitored accompanying chemical induction of domes in a clonal subline of MDCK kidney epithelial cells. Confluent cell monolayers grown on nitrocellulose filters exhibited a relatively high mean transepithelial resistance (387 ohms · cm²). Hexamethylene bisacetamide, a potent inducer of dome formation (Lever, 1979b), stimulated significantly increased transmonolayer resistance as well as elevated levels of intracellular cyclic AMP. By contrast, dimethylformamide, an equally potent inducer of dome formation in MDCK cells, did not appreciably alter either resistance values or cyclic nucleotide levels. These results suggest that induction of dome formation in epithelial cell cultures by compounds generally known as inducers of differentiation may involve multiple and separate mechanisms.     

Lysosomotropic agents and inhibitors of cellular transglutaminase stimulate dome formation, a differentiated characteristic of MDCK kidney epithelial cell cultures

Dome formation is a manifestation of transepithelial fluid transport in cell culture, a differentiated characteristic of transporting epithelia. A dramatic increase in numbers of domes in confluent MDCK kidney epithelial cell cultures was noted after addition of Friend cell inducers such as hexamethylane bisacetamide (HMBA) (Lever, 1979b). In the present study, we show that primary amines such as methylamine, ethylamine, and dansyl cadaverine also stimulate dome formation. These compounds largely prevented the marked decrease in numbers of spontaneously occurring domes which occurred when cultures were switched from medium containing 10% serum to medium containing serum concentrations below 0.2%. Many of these primary amines are not only lysosomotropic agents but also potent inhibitors of transglutaminase activity when assayed in MDCK cell extracts, at concentrations correlating with those effective in stimulation of dome formation. Other lysosomotropic agents such as chloroquine and secondary and tertiary amines stimulated dome formation yet did not inhibit transglutaminase. Induction of domes by HMBA differed in several properties from that stimulated by amines and did not involve fluctuations in transglutaminase activity. These findings suggest that lysosomal functions modulate serum stimulation of dome formation in epithelial cells by a pathway distinct from that triggered by HMBA.     

Effect of tunicamycin on polarized membrane functions of an established kidney epithelial cell line

We have explored the relationship between glycoprotein biosynthesis, cell proliferation and function of a differentiated polarized membrane assessed by dome formation in the MDCK epithelial cell line. At 0.1 μg/ml tunicamycin, complete inhibition of cell proliferation was observed in either serum-containing or serum-free, hormone-supplemented growth medium. By contrast, no inhibition of either spontaneous dome formation or that triggered by inducers of cell differentiation such as hexamethylene bisacetamide was observed at 0.5 μg/ml tunicamycin, although total glycosylation of cellular proteins was inhibited by 75%. Our results suggest that the polarized sorting out of epithelial membrane proteins to apical and basolateral surfaces and their functions related to vectorial transepithelial fluid transport, monitored by dome formation, can persist umimpaired despite considerable underglycosylation of cellular glycoproteins and inhibition of cell proliferation.     

Gangliosides stimulate dome formation in cultured canine kidney epithelial cell line (MDCK)

The effect of exogenous gangliosides on the occurrence of domes in MDCK cell cultures was investigated in view of the involvement of both dome formation and gangliosides in cell growth, differentiation and transepithelial transport. Dome formation was increased by gangliosides in medium free of fetal calf serum. Among the gangliosides tested, GM3 and GD3 isolated from porcine kidney were most active, increasing the dome number 12-17-fold. Since gangliosides from kidney were more active than those from brain and erythrocytes, the hydrophobic moiety as well as sialic acid might be involved in this activity. These results indicate that tissue-specific molecules of gangliosides function as inducers or mediators of dome formation. The mechanism probably involves adenylate-cyclase or another transmembrane biosignal-transducing system.     

The bone morphogenetic protein receptor-1A pathway is required for lactogenic differentiation of mammary epithelial cells in vitro

Bone morphogenetic proteins (BMPs) have been implicated in the control of proliferation, tissue formation, and differentiation. BMPs regulate the biology of stem and progenitor cells and can promote cellular differentiation, depending on the cell type and context. Although the BMP pathway is known to be involved in early embryonic development of the mammary gland via mesenchymal cells, its role in later epithelial cellular differentiation has not been examined. The majority of the mammary gland development occurs post-natal, and its final functional differentiation is characterized by the emergence of alveolar cells that produce milk proteins. Here, we tested the hypothesis that bone morphogenetic protein receptor 1A (BMPR1A) function was required for mammary epithelial cell differentiation. We found that the BMPR1A-SMAD1/5/8 pathway was predominantly active in undifferentiated mammary epithelial cells, compared with differentiated cells. Reduction of BMPR1A mRNA and protein, using short hairpin RNA, resulted in a reduction of SMAD1/5/8 phosphorylation in undifferentiated cells, indicating an impact on this pathway. When the expression of the BMPR1A gene knocked down in undifferentiated cells, this also prevented beta-casein production during differentiation of the mammary epithelial cells by lactogenic hormone stimulation. Addition of Noggin, a BMP antagonist, also prevented beta-casein expression. Together, this demonstrated that BMP-BMPR1A-SMAD1/5/8 signal transduction is required for beta-casein production, a marker of alveolar cell differentiation. This evidence functionally identifies BMPR1A as a potential new regulator of mammary epithelial alveolar cell differentiation.     

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG^−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.     

Proliferation and differentiation of Xenopus A6 cells under hypergravity as revealed by time-lapse imaging

Xenopus laevis A6 cells, which are cloned epithelial cells from the Xenopus kidney, differentiate into a dome structure when the cells reach confluence. We investigated the gravitational responses of A6 cellular motility during normal differentiation and differentiation under hypergravity conditions using centrifugation (1-100 x g). Progression to dome formation was analyzed by time-lapse micrography. Dome formation and increased expression of Na(+)/K(+)-adenosine triphosphatase were used as markers of differentiation. Interestingly, a high rate of cellular proliferation was observed at a low level of hypergravity (5 x g). Despite this, there was no difference in the time to dome formation between the control cells at primary cell density and those that differentiated under hyper- or hypogravity conditions. In conclusion, this experiment on amphibian cells revealed that the proliferation of A6 cells was strongly affected by gravity conditions, but the differentiation step appears to be controlled by an intra- or intercellular clock.     

Cell Organization and Responsiveness to Hormones in Vitro: Genesis of Domes in Mammary Cell Cultures

Domes are multicellular structures generated from confluentmonolayers of mammary epithelium under the influence of insulinand a corticosteroid hormone. The hemicyst structure and occurrencepatterns of domes suggest an in vitro analogy to organized aciniof mammary parenchyma. Two activities of dome cells, vegetativereplication of the mammary tumor virus and synthesis of casein,suggest a functional analogy between domes and acini. The corticosteroidhormone is considered the primary hormonal stimulant for domeformation. Evidence is presented that RNA and protein synthesisis required for the corticosteroid effect, as are intact activetransport functions of epithelial cells.     

DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium

Alterations in gene expression accompany cell-type-specific differentiation. In complex systems where functional differentiation depends on the organization of specific cell types into highly specialized structures (tissue morphogenesis), it is not known how epigenetic mechanisms that control gene expression influence this stepwise differentiation process. We have investigated the effect of DNA methylation, a major epigenetic pathway of gene silencing, on the regulation of mammary acinar differentiation. Our in vitro model of differentiation encompasses human mammary epithelial cells that form polarized and hollow tissue structures (acini) when cultured in the presence of basement membrane components. We found that acinar morphogenesis was accompanied with chromatin remodeling, as shown by alterations in histone 4 acetylation, heterochromatin 1 protein, and histone 3 methylated on lysine 9, and with an increase in expression of MeCP2, a mediator of DNA-methylation-induced gene silencing. DNA hypomethylation induced by treatment with 5-aza-2' deoxycytidine during acinar differentiation essentially prevented the formation of apical tissue polarity. This treatment also induced the expression of CK19, a marker of cells that are in a transitional differentiation stage. These results suggest that DNA methylation is a mechanism by which mammary epithelial differentiation is coordinated both at the tissue and cellular levels.     

Regulation of Na+, K+-ATPase activity in MDCK kidney epithelial cell cultures: Role of growth state,cyclic AMP,and chemical inducers of dome formation and differentiation

Na⁺,K⁺-ATPase activity was monitored in MDCK kidney epithelial cell monolayers and in cell extracts as a function of cell density, cAMP elevation, and exposure to hexamethylene bisacetamide (HMBA) and dimethylsulfoxide (Me₂SO). Ouabain-sensitive Na⁺,K⁺-ATPase and ⁸⁶Rb⁺ uptake activities, and the number of [³H]-ouabain binding sites were maximal in subconfluent cultures and decreased accompanying the development of a confluent monolayer. A sodium pump density of 8 × 10⁷ pumps/cell was estimated for subconfluent cultures, declining to 9 × 10⁵ pumps/cell at confluence. Previous studies have shown that dibutyryl cyclic AMP (Bt₂cAMP), 1-methyl-3-isobutylxanthine (IBMX), or the differentiation inducers HMBA and Me₂SO, which also caused cAMP elevation, all stimulated dome formation, a visible manifestation of active transepithelial Na⁺ and water transport (Lever, 1979). In the present study, all of these inducers were found to elevate intracellular Na⁺ content, implicating this variable in control of induction of dome formation. Operationally, inducers could be divided into two classes. HMBA and Me₂SO partially inhibited ouabain-sensitive ⁸⁶Rb⁺ influx. Ouabain, at a concentration that caused partial sodium pump inhibition and increased intracellular Na⁺ content, was also effective as an inducer. The second class, exemplified by IBMX and Bt₂cAMP caused a furosemide-sensitive increase in intracellular Na⁺ content. This class of inducers stimulated ouabain-sensitive ⁸⁶Rb⁺ uptake, presumably by substrate effects due to increased Na⁺ levels. The Na⁺ or ATP activation of Na⁺,K⁺-ATPase activity assayed in cell-free extracts, the affinity of the transport system for Rb⁺ in intact cells and intracellular ATP levels were unchanged by inducer treatment. Elevation of intracellular Na⁺ concentration, either by cAMP-stimulated, furosemide-sensitive mechanisms or by partial inhibition of the sodium pump may stimulate the induction of dome formation in MDCK cells.     

Analysis of erythroid differentiation in Friend cells using noninducible variants

A series of Friend cell variants has been isolated by selecting for resistance to different inducers of Friend cell differentiation. This procedure selects for cells which have lost the capacity to differentiate terminally in the presence of inducer. Fluctuation analysis shows that these variants arise during culture and are not induced by the selective conditions. Moreover, mutagenesis of parental cells increases the frequency of occurrence of DMSO-resistant variants. Our evidence suggests that these resistant variants arise by two mechanisms. Some arise spontaneously at a relatively high rate (5 × 10^?5?5 × 10^?6 per cell per generation), but their phenotypes are not necessarily stable on removal of the selective conditions. Stable variants arise spontaneously at a lower frequency which is consistent with a true mutational origin.Screening of these stable resistant variants shows that they have different phenotypes. Some fail to respond to any inducer; others respond to all inducers tested except the one used for selection, whereas others respond to some but not all inducers. Most of the DMSO-resistant variants are noninducible by DMSO for all aspects of Friend cell differentiation tested (that is, globin mRNA, hemoglobin, spectrin and the ability to undergo terminal differentiation). Two variants, however, are inducible for an early marker of differentiation, the erythrocyte membrane protein spectrin, but not for other markers such as hemoglobin, globin RNA or terminal differentiation. This implies that the regulation of the globin pathway can be uncoupled from that of spectrin.     

Ionic regulation of MEL cell commitment

A key event in the initiation of the dimethyl sulfoxide (DMSO)-induced program of murine erythroleukemia (MEL) cell differentiation is a rise in the level of cytoplasmic calcium ions. Our interest in the present study is whether other inducers of the terminal erythroid differentiation program also act via a calcium-dependent pathway. Inhibition of calcium transport has been found to prevent the induction of MEL cell commitment by DMSO, butyric acid (BA), or hypoxanthine (HX). Enhancement of the calcium flux rate with A23187 or elevation of cytoplasmic calcium levels with FCCP stimulates the kinetics of commitment in response to all three inducers. These results suggest that of the inducers we have tested (DMSO, BA, and HX), all three act to initiate commitment via a common mechanism which involves modulation of cytoplasmic calcium levels.     

Differentiation-inducing agents decrease cryptic prolactin receptors in cultured rat mammary tumor cells

Normal proliferating and neoplastic mammary cells in culture have cryptic prolactin receptors. These cryptic sites represent 80-95% of the total receptors and can be unmasked by energy depletion. Since lactating mammary tissue and other prolactin targets do not contain cryptic receptors, we have suggested that these sites may be important in the growth response to prolactin. In this study, therefore, we determined the effects of dimethylsulfoxide (DMSO) and sodium butyrate, two inducers of differentiation in other cell systems, on primary cultures of 7,12-dimethylbenzanthracene-induced rat mammary tumors. These substances decreased cryptic receptor levels and inhibited growth. Sodium butyrate (5 mM) decreased receptor levels within 3 h; by 24 h, receptor levels averaged 11 +/- 3% of the controls (n = 13). Similarly, DMSO (1-5%) caused a dose-dependent decrease in receptor levels. With 4% DMSO, there was a progressive decrease in prolactin binding to a nadir of 22 +/- 6% of the controls (n = 8) at 12-24 h. Receptor levels returned to pretreatment values by 24 h after the removal of sodium butyrate or DMSO. In addition, sodium butyrate and DMSO increased the formation of the multicellular structures called 'domes' and the accumulation of lipid droplets. Since sodium butyrate and DMSO decreased cryptic sites, inhibited cell growth and evoked the expression of some morphologic features of differentiation, we conclude that the loss of cryptic prolactin receptors may be involved in the acquisition of a differentiated phenotype in mammary cells.     

Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase.

Mouse myeloid leukemic line M1 cells can be induced to differentiate into the monocyte/macrophage pathway by various inducers. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation inhibiting factors (I-factors) in a cell lysate and conditioned medium of differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have now purified one of the I-factors from conditioned medium of differentiation resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16000-17000 Da (16K I-factor). The amino acid sequence of all fragments of the 16K I-factor we have found are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor, a candidate suppressor protein for differentiation of leukemic cells, is Nm23/nucleoside diphosphate kinase protein.     

Restrictins: stromal cell associated factors that control cell organization in hemopoietic tissues

Hemopoiesis is a sequence of events initiated by the self-renewal of pluripotent stem cells followed by a series of differentiation steps and completed in the formation of distinct tissue patterns. Differentiation and self-renewal are antagonistic processes. A mechanism that attenuates the differentiation flow is obligatory to prevent the exhaustion of the stem cell pool. We suggest that stromal cells from the bone marrow control stem cell renewal through a mechanism that does not require colony-stimulating factors. The organization of cells within the tissue and their specific localization is suggested to be directed by stromal cell activities other than differentiation inducers. These stromal cell activities restrict differentiation or accumulation of mature cells. They are therefore designated as 'Restrictins'.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lactation-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.