Correlation between nuclear histone acetylation and casein messenger RNA induction in the mammary gland

Sodium butyrate prevented the accumulation of casein mRNA induced by the combined action of prolactin and glucocorticoid in the presence of insulin in the cultured mammary gland. This inhibition was reversible and dose-dependent. In addition to the inhibition of the mRNA induction, both nuclear histone acetylase and deacetylase activities were inhibited by the incubation of the glands with butyrate, whereas these enzyme activities were stimulated by glucocorticoid and prolactin, or by glucocorticoid alone, in the presence of insulin. These data strongly suggest that the increased metabolism of histone acetyl groups is involved in the hormone-mediated casein mRNA induction and that glucocorticoid plays a preferential role on this increased metabolism.     

An essential role for glucocorticoid in casein gene expression in rat mammary explants

It is demonstrated that the accumulation of 42 K casein mRNA in mammary tissue from adrenalectomized, virgin rats is almost 20x higher in the presence of exogenous hydrocortisone than in its absence. Accumulation of 25 K casein mRNA in this tissue is totally dependent on the steroid. The results indicate a much greater dependency on hydrocortisone than was appreciated previously, and also show that this dependency does not reflect a loss of cell viability in the absence of the steroid.     

Induction of osteopontin gene expression during mammary gland involution and effects of glucocorticoid on its expression in mammary epithelial cells

To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HC11 cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.     

Characterization of a glucocorticoid receptor in neonatal rat mammary gland

A glucocorticoid receptor has been identified in cytosolic fractions prepared from 4-day old female Sprague-Dawley rat mammary glands at an early resting stage of mammary development. This component sedimented at 10S and 5S on respectively low and high (0.4 M KCl) ionic strength gradients. It bound dexamethasone with a high affinity (Kd approximately 2-6 nM) and a low capacity (N = 300 +/- 100 fmol per mg of proteins or 3.3 +/- 1.3 fmol per micrograms DNA), with a hierarchy of affinity by competition studies dexamethasone greater than corticosterone greater than progesterone greater than R 5020 much greater than Estradiol-17 beta. The characteristics of this glucocorticoid-binding protein are thus very similar to the adult one isolated from adult rat mammary gland.     

Lipotropes alter casein gene expression in bovine mammary acinar culture

Lipotropes (methyl group containing nutrients, including methionine and choline, folic acid, and vitamin B(12)) are essential for cell proliferation and differentiation of mammary tissues. Lipotropes interact in the supply and regulation of intracellular methyl group pools, thereby affecting synthesis and methylation of DNA. To determine the effect of lipotropes on milk protein gene expression, acini isolated from mammary tissues of lactating cows were cultured in one of three treatment media: (1) control, (2) lipotrope deficient, and (3) lipotrope supplemented. beta-Casein mRNA was determined by Northern blotting, and milk protein secretion was measured by a pulse-chase method. The level of beta-casein mRNA was lower in cells grown in lipotrope-deficient medium than in cells grown in the lipotrope-supplemented and control media. Acinar cells cultured in lipotrope-deficient medium also had approximately threefold less milk protein secretion than that of cells in either control or lipotrope-supplemented media. Protein secretion did not differ in the control and lipotrope-supplemented groups. The present study indicates that lipotrope deficiency suppresses total protein secretion and beta-casein gene expression in bovine mammary alveolar epithelial cells in culture.     

Purification and tissue-specific expression of casein kinase from the lactating guinea-pig mammary gland

A serine-specific casein kinase, an integral membrane protein of the lactating guinea-pig mammary gland, has been purified from a Golgi-enriched membrane fraction, using a combination of sucrose gradient centrifugation and chromatography on ATP-agarose. The enzyme comprises a polypeptide of estimated Mr 74 000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, compared with a monomer Mr of 50 000 as determined by sucrose gradient centrifugation in the presence of 500 mM NaCl and 0.1% Triton X-100. Kinetic studies show that the purified enzyme exhibits kinetic constants distinctly different from the rabbit reticulocyte casein kinases I and II, whilst polyclonal antisera raised against the mammary gland enzyme did not cross-react with soluble liver or reticulocyte protein kinase activities. Immunoblotting and immunocytochemical analyses demonstrate the mammary gland enzyme's apparently unique location in lactating mammary gland tissue. Comparative studies with polyclonal antisera raised against bovine galactosyltransferase, show that casein kinase and galactosyltransferase have a similar intracellular localisation in the lactating mammary gland as judged by immunocytochemistry at the light level, but that casein kinase was unique to mammary gland whereas galactosyltransferase could be found in other tissues. The results extended our earlier observations which suggest a Golgi location for casein kinase, and demonstrate that future studies using this enzyme may well prove advantageous for the study of intracellular mechanisms involved in the biogenesis of organelles, in this instance the Golgi apparatus.     

IFN-gamma mediates increased glucocorticoid receptor expression in murine macrophages.

Exposure of the murine macrophage cell line, RAW 264.7, to murine rIFN-gamma resulted in a significant increase in the number of glucocorticoid receptors (GcR). A doubling in the number of GcR was observed as early as 24 h after rIFN-gamma treatment, and receptor number was maximal by 36 h after rIFN-gamma treatment and represented approximately a fourfold increase. Scatchard analysis indicated that a twofold increase in GcR affinity was concomitant with the rIFN-gamma-induced increase in GcR number in RAW 264.7 cells. Increased GcR numbers were induced after exposure of RAW 264.7 cells to as little as 0.1 U/ml rIFN-gamma, and optimal expression was observed at 5 U/ml. Treatment of peritoneal exudate macrophages from C3H/OuJ mice and the LPS hyporesponsive mouse strain, C3H/HeJ, with rIFN-gamma induced an approximately twofold increase in the GcR with no concomitant change in receptor affinity. These results suggest that IFN-gamma may be essential not only for macrophage activation, but also for increasing macrophage sensitivity to feedback inhibition by glucocorticoids by increasing the number and/or affinity of available GcR.     

Corrigendum - Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice

Transgenic Research -     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth in vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogen- or prolactin-dependent growth. Primary tumors still displayed a constitutional expression of alpha-, beta-, and gamma-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in alpha- and beta-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of alpha-, beta-, and gamma-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10-14 days. The accumulation of beta-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance beta-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Cellular localization and distribution of glucocorticoid receptor immunoreactivity and the expression of glucocorticoid receptor messenger RNA in rat pituitary gland

By means of double immunohistochemical techniques and a nonradioisotopic in situ hybridization method, we determined the colocalization pattern of glucocorticoid receptor (GR) and pituitary hormones and the GR messenger RNA (mRNA) expression in the pituitaries of Wistar adult male rats. Immunoreactivity for GR was detected in the nuclei of cells in the anterior and posterior pituitary. Double immunohistochemistry revealed that the colocaliza- tion of GR and anterior pituitary hormones occurred in almost 99% of the growth hormone (GH)-producing cells and adrenocorticotropic hormone (ACTH)-producing cells, and in 67% of the thyroid stimulating hormone (TSH)-producing cells. Almost all of the folliculostellate cells (93%), marginal layer cells (94%) in the anterior pituitary, and pituicytes (96%) in the posterior pituitary immunostained for S100 protein antibody were also immunostained with GR. GR mRNA was abundant in the cytoplasm of anterior and intermediate pituitary cells but scattered sparsely in that of the posterior pituitary. These results suggest that glucocorticoids directly influence certain pituitary cells in order to regulate cell function, including the synthesis and/or secretion of hormones.     

Diet-induced obesity impairs mammary development and lactogenesis in murine mammary gland

We have developed a mouse model of diet-induced obesity that shows numerous abnormalities relating to mammary gland function. Animals ate approximately 40% more calories when offered a high-fat diet and gained weight at three times the rate of controls. They exhibited reduced conception rates, increased peripartum pup mortality, and impaired lactogenesis. The impairment of lactogenesis involved lipid accumulation in the secretory epithelial cells indicative of an absence of copius milk secretion. Expression of mRNAs for beta-casein, whey acid protein, and alpha-lactalbumin were all decreased immediately postpartum but recovered as lactation was established over 2-3 days. Expression of acetyl-CoA carboxylase (ACC)-alpha mRNA was also decreased at parturition as was the total enzyme activity, although there was a compensatory increase in the proportion in the active state. By day 10 of lactation, the proportion of ACC in the active state was also decreased in obese animals, indicative of suppression of de novo fatty acid synthesis resulting from the supply of preformed fatty acids in the diet. Although obese animals consumed more calories in the nonpregnant and early pregnant states, they showed a marked depression in fat intake around day 9 of pregnancy before food intake recovered in later pregnancy. Food intake increased dramatically in both lean and obese animals during lactation although total calories consumed were identical in both groups. Thus, despite access to high-energy diets, the obese animals mobilized even more adipose tissue during lactation than their lean counterparts. Obese animals also exhibited marked abnormalities in alveolar development of the mammary gland, which may partially explain the delay in differentiation evident during lactogenesis.