Synthesis of calelectrins and calpactin I during cytochalasin mediated cell spreading inhibition

Mammary epithelial cell spreading on collagen gels has previously been shown to be correlated with the synthesis of a group of calcium-binding proteins (CBPs) which we have identified as the calcium-binding proteins termed calelectrins and calpactin I monomer/p36. To determine whether cell spreading per se is required for CBP synthesis, we examined the effect of cytochalasin D on these two events. Concentrations of cytochalasin D that did not reduce total protein synthesis, caused inhibition of cell spreading in a dose-dependent manner, but did not cause inhibition of CBP synthesis. Synthesis of collagen also continued during cytochalasin inhibition of cell spreading. Removal of the inhibitor from the cultures initiated cell spreading and CBP synthesis continued. Membrane-cytoskeleton complexes from control and CD treated cells were identical in regard to binding CBPs in a calcium-dependent manner. Colchicine, which inhibited cell spreading, was shown to be toxic to general protein synthesis at 75 nM. The data clearly indicate that mere inhibition of epithelial cell spreading does not automatically suppress CBP synthesis.     

Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dose-dependent manner. Cytochalasin D inhibition of F-actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.     

Synthesis of novel calcium-dependent proteins associated with mammary epithelial cell migration and differentiation

A class of proteins from mouse mammary epithelial cells has been isolated which, like the calcium-binding protein calmodulin (CaM), binds to phenothiazine in a calcium-dependent manner. These proteins do not bind to phenothiazine through binding to CaM; we infer that they are calcium-binding proteins, and that they may be related to the similarly isolated 'calcimedins' of Moore, P D & Dedman, J, J biol chem 257 (1982) 9663 [8]. In primary cultures of mouse mammary cells on collagen gels, synthesis of certain of these proteins is associated with the spreading of cells to form monolayers; failure of cells to spread and differentiate, through omission of serum from culture medium, results in the inhibition of calcium-binding protein synthesis, with the exception of CaM and a 15 kD species. The CaM/15 kD pair are prominent during all phases of culture, and are secreted during the secretory differentiation phase of culture (floating gels). We propose that these calcium-binding proteins play a specific role in the motility of mammary epithelial cells and that they may also be involved in mammary secretory differentiation.     

The collagen recognition sequence for fibroblasts depends on collagen topography

We found that the peptide Gly-Arg-Gly-Glu-Ser-Pro (GRGESP) inhibited spreading of human fibroblasts inside collagen gels and markedly decreased gel contraction, but this peptide had no effect on cell spreading on collagen-coated surfaces. On the other hand, the peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), which inhibited cell spreading on collagen-coated surfaces, did not inhibit cell spreading within collagen gels and was a less effective inhibitor of collagen gel contraction than GRGESP. Based on these findings, we conclude that human fibroblasts can interact with different collagen cell recognition sequences depending upon topographical organization of the collagen.     

Basement membrane collagen requirements for attachment and growth of mammary epithelium.

In vivo mammary epithelial cells rest upon a basement membrane composed in part of type IV collagen which is synthesized by these cells. In this study, basement membrane collagen is shown to be selectively recognized by normal mammary ducts and alveoli for attachment and growth when compared to the types of collagen derived from stroma (types I or III) or cartilage (type II). Cell attachment and growth on type I collagen is inhibited by the proline analogue, cis-hydroxyproline, which blocks normal collagen production. These effects of cis-hydroxyproline are not apparent when a basement membrane collagen substratum is provided. Unlike normal mammary epithelium, mammary fibroblasts show little preference for the collagen to which they will attach. A requirement of type IV collagen synthesis for normal mammary epithelial cell attachment and growth on stromal collagen in vitro may have significance in vivo where a basement membrane scaffold may be necessary for normal mammary morphogenesis and growth.     

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Collagen processing in ras-transfected mouse mammary epithelial cells

A mouse mammary epithelial cell line (NMuMG), after transfection with the c-rasH oncogene, forms invasive tumors in nude mice. NMuMG and NMuMG/p-rasH cells produce similar amounts of collagen (mostly type IV) when grown on plastic. NMuMG cells respond to growth on collagen gels by increasing the rate of collagen synthesis and deposition by 100%, unlike NMuMG/p-rasH cells which synthesize similar amounts of collagen whether grown on plastic or collagen gels. These results suggest that ras transformation partially inhibits the interaction between epithelial cells and the surrounding stroma that is necessary for basement membrane deposition in vivo and consequently may facilitate the invasion of the stroma by transfected cells.     

p130cas but not paxillin is essential for Caco-2 intestinal epithelial cell spreading and migration on collagen IV

We have previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src kinase and stimulates Src-dependent tyrosine phosphorylation of p130cas. We observed that collagen IV also stimulated Src-dependent phosphorylation of both paxillin Tyr31 and paxillin Tyr118. Caco-2 transfection with paxillin or p130cas siRNAs inhibited expression of these proteins by more than 90% for at least 5 days after transfection. Although p130cas siRNA inhibited cell spreading on collagen IV by 33%, three different paxillin siRNAs did not inhibit cell spreading. p130cas siRNA did not affect Src Tyr416 or Src Tyr527 phosphorylation, FAK Tyr397 phosphorylation, or Src-dependent phosphorylation of FAK Tyr925, suggesting that p130cas did not inhibit cell spreading by altering FAK or Src activity. Rat p130cas expression after siRNA knock-out of endogenous human p130cas in Caco-2 cells reduced cell spreading inhibition by 71%. In contrast, expression of rat p130cas from which the Src-phosphorylated substrate domain was deleted did not rescue siRNA inhibition of cell spreading. Combined treatment with siRNAs to Crk and CrkL, which bind to the p130cas substrate domain, inhibited cell spreading by 54%. Both p130cas siRNA and the combined Crk/CrkL siRNAs strongly inhibited (52 and 46% inhibition, respectively) Caco-2 sheet migration on collagen IV and noticeably inhibited lamellipodial extension, whereas paxillin siRNA only inhibited migration by 18% and did not noticeably affect lamellipodial extension. These results suggest that Src may regulate Caco-2 migration on collagen IV via both p130cas and paxillin but that Src phosphorylation of p130cas is more important for this process.     

Collagen IV regulates Caco-2 migration and ERK activation via alpha1beta1- and alpha2beta1-integrin-dependent Src kinase activation

Our previous work indicates intestinal epithelial cell ERK activation by collagen IV, a major component of the intestinal epithelial basement membrane, requires focal adhesion kinase (FAK) and suggests FAK and ERK may have important roles in regulating intestinal epithelial cell migration. We therefore sought to identify FAK downstream targets regulating intestinal epithelial cell spreading, migration, and ERK activation on collagen IV and the integrins involved. Both dominant-negative Src and Src inhibitor PP2 strongly inhibited collagen IV ERK activation in Caco-2 intestinal epithelial cells. Collagen IV stimulated Grb2 binding site FAK Y925 phosphorylation, which was inhibited by PP2 and required FAK Y397 autophosphorylation. Additionally, FAK Y925F expression blocked collagen IV ERK activation. alpha(1)beta(1)- Or alpha(2)beta(1)-integrin blockade with alpha(1)- or alpha(2)-integrin subunit antibodies indicated that either integrin can mediate adhesion, cell spreading, and FAK, Src, and ERK activation on collagen IV. Both dominant-negative Src and PP2 inhibited Caco-2 spreading on collagen IV. PP2 inhibited p130(Cas) tyrosine phosphorylation, but dominant-negative p130(Cas) did not inhibit cell spreading. PP2 inhibited Caco-2 migration on collagen IV much more strongly than the mitogen-activated protein kinase kinase inhibitor PD-98059, which completely inhibited collagen IV ERK activation. These results suggest a pathway for collagen IV ERK activation requiring Src phosphorylation of FAK Y925 not previously described for this matrix protein and suggest either alpha(1)beta(1)- or alpha(2)beta(1)-integrins can regulate Caco-2 spreading and ERK activation on collagen IV via Src. Additionally, these results suggest Src regulates Caco-2 migration on collagen IV primarily through ERK-independent pathways.     

Extracellular matrix control of collagen production by rat mammary epithelial and myoepithelial-like cells in vitro

A rat mammary myoepithelial cell line (Rama 401) grown on plastic produces 5 times more collagen (largely type IV) than a mammary epithelial cell line (Rama 704) grown on the same surface. When the cells are grown on collagen gels, the amount of collagen produced by Rama 704 cells increases 3.3 times, whereas there is no increase in collagen production by Rama 401 cells. Increased production of collagen by Rama 704 cells is due to both an increased rate of synthesis and a decreased rate of degradation. These results indicate that for mammary epithelial cells, unlike myoepithelial cells, the rate of production of collagen can be regulated by the extracellular matrix.     

Correlation of tumorigenicity with resistance to growth inhibition by cis-hydroxyproline

cis-Hydroxyproline, an inhibitor of collagen deposition, was examined for its effect on the growth of a variety of tumorigenic and non-tumorigenic cells. Virally, chemically, and spontaneously transformed murine cell lines were found to be less sensitive to cell spreading and growth inhibition by cis-hydroxyproline (CHP) than were their non-tumorigenic counterparts. The non-tumorigenic lines exhibited a higher rate of collagen accumulation in culture than the tumorigenic cell lines. The rate of collagen accumulation in culture without CHP and the growth inhibition by CHP were directly related. These results suggest that normal but not tumorigenic cells may require synthesis of an extracellular substrate containing collagen to support spreading and growth.     

HH2A,an immortalized bovine mammary epithelial cell line,expresses the gene encoding mammary derived growth inhibitor (MDGI)

Summary We have established and partially characterized a spontaneously immortalized bovine mammary epithelial cell line, designated HH2a. The cells express the gene encoding for mammary derived growth inhibitor (MDGI) when grown on released collagen gels in the presence of lactogenic hormones. This is the first report of a cell line that expresses MDGI. Immunohistochemical studies showed that HH2a cells contain keratin intermediate filaments and desmosomes. When plated on confluent monolayer of live fibroblasts, HH2a cells extensively contacted with fibroblasts. When embedded in the collagen gels, they rearranged themselves to produce three-dimensional duct-like outgrowths extending into the matrix. The HH2a cell line should be useful in investigations of the roles of cell-cell and cell-extracellular interactions in regulation of breast epithelial cell proliferation, and of the hormonal regulation of MDGI gene expression.     

Morphology and lactose synthesis in tissue culture of mammary alveoli isolated from lactating mice

Summary Mammary epithelial cells from lactating mice synthesize and secrete lactose in culture and retain many features of their in vivo morphology if mammary glands are only partially dissociated to alveoli, rather than completely dissociated to single cells. After 5 d in culture lactose synthesis by alveoli cultured on floating collagen gels is 10 to 20 times higher than in cultures of single cells on floating collagen gels. Moreover, mammary alveoli in culture retain sensitivity to lactogenic hormones; the synthesis of lactose by alveoli depends on the continued presence of insulin and either hydrocortisone or prolactin. In addition, within alveoli the original juxtaposition of constituent epithelial cells is retained, and cells are cuboidal and have many microvilli and fat droplets. In contrast, alveoli on attached gels flatten and lose their secretory morphology. These results indicate that the shape of the cells, presence of lactogenic hormones, and maintenance of epithelial:epithelial cell contacts are required for maintenance of mammary epithelial cell differentiation in culture. This research was supported by Grants CA-16392 and AG-02909 from the National Institutes of Health and Institutional Grant IN 119 from the American Cancer Society.     

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.     

Ultrastructural features of bovine mammary epithelial cells grown on collagen gels

Bovine mammary epithelial cells cultured on floating gels of rat tail collagen showed two principal cell types, columnar and squamous, with ultrastructural features resembling secretory and myoepithelial cells respectively. Cultures of freshly prepared cells released alpha-lactalbumin into the culture medium and in some cases contained fat droplets, although these did not appear to be released. No ultrastructural evidence of casein synthesis was observed. A notable feature was the failure to secrete a continuous basement membrane. Intermediate filaments were present in abundance in squamous epithelial cells.     

Regulation of differentiation and polarized secretion in mammary epithelial cells maintained in culture: extracellular matrix and membrane polarity influences

Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium. When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of beta-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of beta-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the beta-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, beta-casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (Millipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions alpha 1-, alpha 2-, and beta-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of the extracellular matrix on plasminogen activator isozyme activities of human mammary epithelial cells in culture.

Multiple molecular forms of plasminogen activator were detected in normal human mammary epithelial cells in culture. Cells derived from (normal) breast mammoplasty specimens and grown on the surface of collagen gels exhibited three major classes of plasminogen activator isozymes (Mr = 100,000 [100K], 75,000 [75K], and 55,000 [55K]). The activity of the 100K and 75K isozymes was greatly reduced when the cells were grown on conventional tissue-culture-grade plastic surfaces. MCF-7, a human mammary carcinoma cell line, exhibited predominantly or exclusively the 55K isozyme, irrespective of the cell growth substratum. The activity of the 55K isozyme was more than twofold higher for MCF-7 cells grown on collagen gels than for cells grown on plastic. Progesterone, diethylstilbestrol, and estrogen stimulated the activity of the 55K isozyme of MCF-7 cells, but only when the cells were grown on a plastic surface. The plasminogen activator activities of the normal human mammary epithelial cells were not stimulated by these hormones, irrespective of the growth substratum. These results show that the expression of plasminogen activator isozymes by human mammary epithelial cells is subject to modulation by the extracellular matrix. Normal and malignant cells may differ in their responsiveness to these effects.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing of luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasional myoepithelial cells, characterized by myofilaments and plasmalemmmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over TO values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes.     

Extracellular matrix organization modulates fibroblast growth and growth factor responsiveness

To learn more about the relationship between extracellular matrix organization, cell shape, and cell growth control, we studied DNA synthesis by fibroblasts in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached 22 mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and fibroblasts had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and fibroblasts had a stellate morphology. DNA synthesis by fibroblasts in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, growth of fibroblasts in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of fibroblasts in contracted collagen gels and monolayer culture to peptide growth factors including fibroblast growth factor, platelet-derived growth factor, transforming growth factor-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the growth factors. Cells in attached collagen gels and monolayer culture were affected similarly by fibroblast growth factor but not by the others. Our results indicate that extracellular matrix organization influenced not only cell growth, but also fibroblast responsiveness to peptide growth factors.