Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice

Whey acidic protein (WAP) is an abundant rodent milk protein. Its expression in mouse mammary epithelial cell cultures was previously found to require the formation of an extracellular matrix (ECM)-induced three-dimensional alveolar structure. In the absence of such structures, cells were shown to secrete diffusible factors leading to suppression of WAP expression. We demonstrate here that (a) TGF-alpha production and secretion by mammary cells is downregulated by the basement membrane-dependent alveolar structure, and (b) compared with beta-casein, WAP expression is preferentially inhibited both in culture and in transgenic mice when TGF-alpha is added or overexpressed. Thus, (c) the enhanced TGF-alpha production when cells are not in three- dimensional structures largely accounts for the WAP-inhibitory activity found in the conditioned medium. Since this activity can be abolished by incubating the conditioned medium with a function blocking antibody to TGF-alpha. The data suggest that ECM upregulates WAP by downregulating TGF-alpha production. We also propose that changes in TGF-alpha activity during mouse gestation and lactation could contribute to the pattern of temporal expression of WAP in the gland. These results provide a clear example of cooperation among lactogenic hormones, ECM, and locally acting growth factors in regulation of tissue-specific gene expression.     

Overexpression of Mos, Ras, Src, and Fos inhibits mouse mammary epithelial cell differentiation.

Mammary epithelial cells terminally differentiate in response to lactogenic hormones. We present evidence that oncoprotein overexpression is incompatible with this hormone-inducible differentiation and results in striking cellular morphological changes. In mammary epithelial cells in culture, lactogenic hormones (glucocorticoid and prolactin) activated a transfected beta-casein promoter and endogenous beta-casein gene expression. This response to lactogenic hormone treatment was paralleled by a decrease in cellular AP-1 DNA-binding activity. Expression of the mos, ras, or src (but not myc) oncogene blocked the activation of the beta-casein promoter induced by the lactogenic hormones and was associated with the maintenance of high levels of AP-1. Mos expression also increased c-fos and c-jun mRNA levels. Overexpression of Fos and Jun from transiently transfected constructs resulted in a functional inhibition of the glucocorticoid receptor in these mouse mammary epithelial cells. This finding clearly suggests that glucocorticoid receptor inhibition arising from oncogene expression will contribute to the block in hormonally induced mammary epithelial cell differentiation. Expression of Src resulted in the loss of the normal organization and morphological phenotype of mammary epithelial cells in the epithelial/fibroblastic line IM-2. Activation of a conditional c-fos/estrogen receptor gene encoding an estrogen-dependent Fos/estrogen receptor fusion protein also morphologically transformed mammary epithelial cells and inhibited initiation of mammary epithelial differentiation-associated expression of the beta-casein and WDNM 1 genes. In response to estrogen treatment, the cells displayed a high level of AP-1 DNA-binding activity. Our results demonstrate that high cellular AP-1 levels contribute to blocking the ability of mammary epithelial cells in culture to respond to lactogenic hormones. This and other studies indicate that the oncogene products Mos, Ras, and Src exert their effects, at least in part, by stimulating cellular Fos and probably cellular Jun activity.     

In vivo and in vitro expression of human serum albumin genomic sequences in mammary epithelial cells with beta-lactoglobulin and whey acidic protein promoters

The expression pattern of human serum albumin (HSA) in transgenic mice carrying various HSA genomic sequences driven either by the mouse whey acidic protein (WAP) or the sheep beta-lactoglobulin (BLG) promoters, was compared. The pattern of HSA expression in both WAP/HSA and BLG/HSA transgenic lines was copy number independent, and the major site of ectopic expression was the skeletal muscle. Although an equal proportion of expressors was determined in both sets of mice (approximately 25% secreting >0.1 mg/ml), the highest level of HSA secreted into the milk in the WAP/HSA transgenic lines was one order of magnitude lower than in the BLG/HSA lines. Despite this difference, the HSA expression patterns in the mammary gland were similar and consisted of two levels of variegated expression. Studies using mammary explant cultures revealed a comparable responsiveness to the lactogenic hormones insulin, hydrocortisone, and prolactin, although the WAP/HSA gene constructs were more sensitive to the hydrocortisone effect than were the BLG/HSA vectors. When HSA vectors were stably transfected into the mouse mammary cell line CID-9, they displayed a hierarchy of expression, dependent upon the specific complement of HSA introns included. Nevertheless, the expression of HSA in four out of five WAP/HSA constructs was similar to their BLG/HSA counterparts. This construct-dependent, and promoter-independent, hierarchy was also found following transfection into the newly established Golda-1 ovine mammary epithelial cell line.     

Loop formation by the transgeneWAP:6xHishGH in transgenic rabbit fibroblasts, revealed by fluorescencein situ hybridization to nuclear halos

Using fluorescence in situ hybridization (FISH) to somatic nuclear halos from transgenic rabbits WAP:6xHishGH, we present evidence for stability of transgenesis at the chromatin level. FISH performed on fibroblasts from a homozygous individual showed 2 independent loops from both chromosomes of pair 7. On a heterozygous individual, FISH detected a single loop. According to the concept of chromatin loops and their influence on gene expression, this shows that the human growth hormone transgene, which was actively expressed in mammary gland under the influence of the tissue-specific promoter, was inactive in examined skin fibroblasts.     

Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation

We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females. PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ(+)) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ(+) cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. LacZ(+) cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ(+) mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse epithelial cell fates.     

Expression of a whey acidic protein transgene during mammary development. Evidence for different mechanisms of regulation during pregnancy and lactation

Expression of the mouse whey acidic protein (WAP) gene is specific to the mammary gland, is induced several thousand-fold during pregnancy, and is under the control of steroid and peptide hormones. To study developmental regulation of the mouse WAP gene, a 7.2-kilobase (kb) WAP transgene, including 2.6 kb of 5'- and 1.6 kb of 3'-flanking sequences, was introduced into mice. Of the 13 lines of mice examined, 6 expressed the transgenes during lactation at levels between 3 and 54% of the endogenous gene. Although expression was dependent on the site of integration, the transgenes within a given locus were expressed in a copy number-dependent manner and were coordinately regulated. The WAP transgenes were expressed specifically in the mammary gland, but showed a deregulated pattern of expression during mammary development. In all six lines of mice, induction of the WAP transgenes during pregnancy preceded that of the endogenous gene. During lactation, expression in two lines increased coordinately with the endogenous gene, and in three other lines of mice, transgene expression decreased to a basal level. These data indicate that the 7.2-kb gene contains some but not all of the elements necessary for correct developmental regulation. At a functional level it appears as if a repressor element, which inactivates the endogenous gene until late pregnancy, and an element necessary for induction during lactation are absent from the transgene. Complementary results from developmental and hormone induction studies suggest that WAP gene expression during pregnancy and lactation is mediated by different mechanisms.     

High-level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3'' untranslated region.

The high-level expression of the rat whey acidic protein (WAP) gene in transgenic mice depends on the interaction of 5'-flanking promoter sequences and intragenic sequences. Constructs containing 949 bp of promoter sequences and only 70 bp of 3'-flanking DNA were expressed at uniformly high levels, comparable to or higher than that of the endogenous gene. Although this WAP transgene was developmentally regulated, it was expressed earlier during pregnancy than was the endogenous WAP gene. Replacement of 3' sequences, including the WAP poly(A) addition site, with simian virus 40 late poly(A) sequences resulted in an approximately 20-fold reduction in the expression of WAP mRNA in the mammary gland during lactation. Nevertheless, position-independent expression of the transgene was still observed. Further deletion of 91 bp of conserved WAP 3' untranslated region (UTR) led to integration site-dependent expression. Position independence was restored following reinsertion of the WAP 3' UTR into the deleted construct at the same location, but only when the insertion was in the sense orientation. The marked differences observed between the expression levels of the 3'-end deletion constructs in transgenic mice were not seen in transfected CID 9 mammary epithelial cells. In these cells, expression of the endogenous WAP gene was dependent on the interaction of these cells with a complex extracellular matrix. In contrast, the transfected WAP constructs were not dependent on extracellular matrix for expression. Thus, both the abnormal expression of WAP in cells cultured on plastic and the precocious developmental expression of WAP in transgenic mice may reflect the absence of a negative control element(s) within these recombinant constructs.     

Milk protein expression and ductal morphogenesis in the mammary gland in vitro: hormone-dependent and -independent phases of adipocyte-mammary epithelial cell interaction

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.