Prolactin receptor expression in the epithelia and stroma of the rat mammary gland

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.     

Epidermal growth factor receptor levels in mouse mammary glands in various physiological states

Experiments were undertaken to demonstrate and characterize specific receptors for epidermal growth factor (EGF) in mammary glands of female BALB/c mice in various physiological states. The results of an in vitro desaturation technique are also presented which allow estimation of the total EGF-binding sites per mg membrane protein. Binding of the ligand [125I]iodo-EGF is both time and temperature dependent. Maximum binding to the membrane is achieved after 6 h of incubation with [125I]iodo-EGF at 23 C. Scatchard analysis of equilibrium binding using membrane preparations of mammary glands from virgin mice yields two classes of high affinity receptors with Kd values of 0.8 +/- 0.1 and 5.0 +/- 0.4 X 10(-10) M and receptor concentrations of 10 +/- 1.2 and 23.5 +/- 2 fmol/mg protein, respectively. Membrane preparations of mammary tissues from cycling, gestating, and lactating mice were used to correlate cellular receptor levels to the physiological state of the animal. Beginning at weaning, there is a constant decrease in high affinity receptor level with increasing age, as well as through the early stages of both gestation and lactation. On day 10 of gestation, receptor levels increase, reaching 15.2 +/- 1.6 fmol/mg protein, followed by a decrease to 3.8 +/- 0.9 fmol/mg protein on day 10 of lactation. We conclude that membrane preparations from the mouse mammary gland contain specific high affinity receptors for EGF, and that receptor levels are characteristic of the physiological state.     

Tissue distribution, characterization, and regulation of messenger ribonucleic acid for growth hormone receptor and serum binding protein in the rat

The distribution of GH receptor (GHR) and GH-binding protein (GHBP) mRNAs in multiple rat tissues was examined by Northern blotting using a cDNA fragment encoding the common extracellular domain of the GHR and the serum GHBP. Both GHR and GHBP mRNAs [4.5 and 1.2 kilobases (kb), respectively] were present in liver, kidney, adrenal, heart, muscle, ovary, mammary gland, gastrointestinal tract, and adipose tissue, but were barely or not detectable in testis, thymus, or brain. These observations suggest that GH exerts direct effects across a broad spectrum of rat tissues. Nuclease protection analysis also confirmed the presence in extrahepatic tissues of a GHR mRNA with up to 50% of the cytoplasmic domain being identical in sequence to that of the hepatic GHR mRNA. This suggests, but does not prove, that different receptor classes with differing intracellular signalling mechanisms may not exist. It is also clear from our studies that liver was the most abundant source of the truncated (1.2 kb) mRNA and is, therefore, believed to be the primary site of GHBP synthesis. Also, more importantly, at least in the rat, it was only GHBP mRNA that was up-regulated to any extent during pregnancy (female vs. pregnant, P less than 0.001). No significant changes were observed in the abundance of the full-length (4.5 kb) GHR mRNA. This pregnancy-related change in GHBP mRNA was also accompanied by a comparable increase in the actual level of measurable serum GHBP (female vs. pregnant, P = 0.005). Little change was seen in hepatic membrane binding. These data suggest that the GHBP and GHR are both widely coexpressed, but that the expression is not always coordinately regulated. This raises the possibility that the GHBP and GHR may have distinct roles in the regulation of GH delivery and action.     

Estrogen treatment in vivo increases keratinocyte growth factor expression in the mammary gland

Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development.     

The effects of ovine placental lactogen and bovine growth hormone on hepatic and mammary gene expression in lactating sheep

The ability of ovine placental lactogen (oPL) to bind to the growth hormone receptor (GHR) raises the possibility that oPL may exert a growth hormone (GH)-like action on galactopoiesis. We have compared the effects of treating lactating ewes for 5 days with an equimolar dose (0.1 mg/kg/day, administered as two equal doses 12 hourly) of either bovine growth hormone (bGH) (n = 10), oPL (n = 10) or saline (n = 9) on hepatic and mammary GHR, insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 (IGFBP-3) gene expression and hepatic GHR number. Hepatic GHR and IGFBP-3 mRNA were unaltered by bGH or oPL treatment. Hepatic IGF-I mRNAs increased following bGH (P < 0.05) but not oPL treatment. GHR gene expression was greater in liver compared to mammary gland extracts. There was no effect of either bGH or oPL treatment on mammary GHR, IGF-I or IGFBP-3 mRNA or hepatic GHR number. These studies confirm the galactopoietic effects of bGH in lactating ruminants and suggest that the mechanism of this action is not via increased hepatic GHR number or gene expression. In addition, the increase in hepatic but not mammary IGF-I mRNA with bGH treatment suggests an endocrine action of IGF-I on milk synthesis. These studies also demonstrate that an equimolar dose of oPL is not galactopoietic or somatogenic in the lactating ewe.     

The role of prolactin and growth hormone in mammary gland development

Development and differentiation of the mammary gland occur primarily during pregnancy. Females homozygous (-/-) for the null mutation of the PRL receptor (PRLR) gene are sterile due to a complete failure of blastocysts to implant. In progesterone-treated mice pregnancy is rescued but the mammary gland is severely underdeveloped. Interestingly, females hemizygous for the PRLR (+/-) in their first lactation show an almost complete failure to lactate. This phenotype disappears in the second and subsequent pregnancies in inbred 129/Sv mice but is maintained in inbred C57BL/6 mice. In GH receptor (GHR) KO mice litter size is markedly decreased, probably due to an ovarian defect. To assess the relevance of the GH and PRLRs in the mammary gland development, GHR and PRLR null epithelia were transplanted into cleared fat pads of wild-type mice. Such studies show that epithelial GHR is not required for functional mammary development. In contrast, epithelial PRLRs are required for mammary development and milk protein gene expression during pregnancy. Since ductal development is impaired in GHR -/- mice, it appears that GH signals through the stromal compartment. In summary, it is now established that GH and PRL activate Stat5 in separate compartments, reflecting their specific roles in ductal and alveolar development and differentiation.     

Acidic and basic fibroblast growth factor mRNA and protein in mouse mammary glands

Previous studies suggest that members of the fibroblast growth factor (FGF) family are mitogenic to mammary epithelium. In order to determine expression of acidic and basic FGF (aFGF and bFGF) during mammary development, mice were euthanized as virgins, early pregnant, mid-pregnant, late-pregnant, or during early lactation. Mammary expression of both aFGF and bFGF mRNA increased through pregnancy. Acidic FGF mRNA continued to increase during early lactation, but basic FGF message level decreased drastically during early lactation. Western blots probed with anti-aFGF showed four immunoreactive bands approx 30, 48, 52, and 55-kDa in size. The 30-, 48-, and 55-kDa bands for aFGF were expressed at low levels during virgin and early pregnant stages but were more prominent during the later stages. The 52-kDa band was high during the virgin and early pregnant stages and low in mid-pregnancy through early lactation. Blots probed with anti-bFGF showed two bands approx 30 and 50 kDa in size. Both bands increased through early-pregnancy, but during late-pregnancy there was a decrease in immunoreactive protein levels, which remained low during early lactation. Experiments to determine where FGF mRNAs are produced in the mammary gland suggest that both FGFs may be produced in the stroma, leading to the hypothesis that aFGF and bFGF are stromally produced growth factors and probably act on the epithelial component of the gland in a paracrine fashion.     

Ontogeny and control of prolactin receptors in the mammary gland and liver of virgin, pregnant and lactating rats.

Prolactin receptors were identified and partially characterized in the mammary gland of the rat. The binding of 125I-labelled ovine prolactin to a subcellular particulate fraction of rat mammary gland decreased between days 30 and 100 of age. Over the same period, binding to the liver increased and there was a significant negative correlation between prolactin binding in the two tissues. Binding to the mammary gland was low during pregnancy, increased in early lactation and declined after the litters were weaned. Binding to the liver was lower during lactation than during pregnancy or the period after weaning suggesting that tissue-specific factors may operate in the control of this receptor. In virgin rats, prolactin binding by the mammary gland was increased by oestrogen. This effect was blocked by hypophysectomy and partially restored by replacement therapy with prolactin. Hypothyroidism and treatment with progesterone also reduced the response to oestrogen. The maintenance of prolactin binding by the mammary gland of lactating rats depends on the presence of the ovaries and pituitary, thyroid and adrenal glands. Examination of the ratio epithelium: stroma suggests that prolactin acts by increasing the number of epithelial cells in the mammary gland and that thyroid, adrenal and ovarian hormones modulate the number of receptors per cell.     

Impaired mouse mammary gland growth and development is mediated by melatonin and its MT1 G protein-coupled receptor via repression of ERα, Akt1, and Stat5

To determine whether melatonin, via its MT(1) G protein-coupled receptor, impacts mouse mammary gland development, we generated a mouse mammary tumor virus (MMTV)-MT1-Flag-mammary gland over-expressing (MT1-mOE) transgenic mouse. Increased expression of the MT(1) -Flag transgene was observed in the mammary glands of pubescent MT1-mOE transgenic female mice, with further significant increases during pregnancy and lactation. Mammary gland whole mounts from MT1-mOE mice showed significant reductions in ductal growth, ductal branching, and terminal end bud formation. Elevated MT(1) receptor expression in pregnant and lactating female MT1-mOE mice was associated with reduced lobulo-alveolar development, inhibition of mammary epithelial cell proliferation, and significant reductions in body weights of suckling pups. Elevated MT(1) expression in pregnant and lactating MT1-mOE mice correlated with reduced mammary gland expression of Akt1, phospho-Stat5, Wnt4, estrogen receptor alpha, progesterone receptors A and B, and milk proteins β-casein and whey acidic protein. Estrogen- and progesterone-stimulated mammary gland development was repressed by elevated MT(1) receptor expression and exogenous melatonin administration. These studies demonstrate that the MT(1) melatonin receptor and its ligand melatonin play an important regulatory role in mammary gland development and lactation in mice through both growth suppression and alteration of developmental paradigms.     

Growth hormone (GH) and 17beta-estradiol regulation of the expression of mouse GH receptor and GH-binding protein in cultured mouse hepatocytes.

In the present study, primary mouse hepatocytes from 8- to 10-week-old virgin female Swiss-Webster mice were perfused with collagenase (100 U/ml) using the two-step method. Isolated hepatocytes were plated in a rat tail type I collagen sandwich configuration to examine the regulation of GH receptor (GHR) and GH-binding protein (GHBP) expression by GH and 17beta-estradiol (E2). After 48 h of initial plating, hepatocytes were divided into groups of five replicates and treated for 24 h with medium containing no hormones (controls), GH (100 ng/ml), E2 (10(-9) M), E2 (10(-9) M) plus GH (100 ng/ml), or E2 plus GH and ICI 182-780 at different concentrations. Treatment of hepatocytes with GH or E2 alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR messenger RNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2 and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. In the present study we have shown that in cultured hepatocytes from virgin female mice, GH or E2 alone did not affect the concentrations of GHBP and GHR. However, E2 and GH together significantly up-regulated GHR and GHBP expression.     

Hormonal regulation of radioiodide uptake activity and Na+/I- symporter expression in mammary glands

The observation that radioiodide uptake (RAIU) activity, mediated by the Na+/I- symporter (NIS), is significantly increased in lactating breast suggests that RAIU and NIS expression in mammary gland are modulated by hormones involved in active lactation. We showed that both the NIS expression level and RAIU in rat mammary gland are maximal during active lactation compared to those in the mammary glands of virgin and pregnant rats as well as the involuting mammary gland. In the lactating mammary gland, NIS is clustered on the basolateral membrane of alveolar cells as a lesser glycosylated form than NIS in thyroid. The RAIU of lactating mammary gland was partially inhibited by treatment with a selective oxytocin antagonist or bromocriptine, an inhibitor of PRL release. These findings suggest that RAIU and NIS expression in mammary gland are at least in part modulated by oxytocin and PRL. Indeed, we showed that NIS messenger ribonucleic acid level was increased in a dose-dependent manner by oxytocin and PRL in histocultured human breast tumors.     

Structure and regulation of expression of the mouse GH receptor

GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes treated with E2 and GH compared with those in untreated cells or cells treated with either E2 or GH alone. Our studies in the mouse suggest that GHBP is an important cell-surface receptor for GH in the liver. These studies postulate that an arginine-glycine-aspartic acid sequence found on mouse GHBP but absent in other species is responsible for the association of GHBP with the plasma membrane by binding to one or more integrins on the surface of liver cells.     

Regulation of the hepatic growth hormone receptor and serum growth hormone-binding protein during pregnancy in the mouse: effects of litter size.

The regulation of hepatic GH receptor (GHR) and serum GH-binding protein (GHBP) during pregnancy in the mouse was investigated by manipulating the number of conceptuses carried by the dam. Animals carrying 1-4 conceptuses had significantly lower amounts of hepatic GHR (GH-binding activity) than mice carrying 10-13 conceptuses on days 9 and 13 of pregnancy. There was no significant difference in hepatic GHR on day 17 of pregnancy between animals carrying 1-4 and 10-13 conceptuses. Animals carrying 1-4 conceptuses had significantly lower concentrations of GHBP in serum than animals carrying 10-13 conceptuses on days 9, 13, and 17 of pregnancy. The relative amounts of liver GHR- and GHBP-encoding messages in animals with low and high conceptus numbers were investigated by Northern analysis. There were higher levels of both messages in animals carrying 10-13 conceptuses than in mice carrying 1-4 conceptuses. On day 13 of pregnancy, animals carrying 10-13 conceptuses had significantly higher levels of GHBP-encoding message than animals carrying 1-4 conceptuses. Total hepatic mass was not significantly different between animals with low and high conceptus numbers. No significant difference was found in GHBP concentration between blood from the uterine vein and the trunk in 17-day pregnant animals. Mouse placental lactogen-I (mPL-I), mPL-II, GH, and corticosterone concentrations were measured by RIA and related to hepatic GHR activity and serum GHBP concentration. Hepatic GHR activity and serum GHBP concentration were significantly correlated with each other on days 9, 13, and 17 of pregnancy. Hepatic GHR activity was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy. GHBP concentration was significantly correlated with mPL-I and mPL-II on day 9 of pregnancy and with mPL-II and GH on day 13 of pregnancy. Data are consistent with the hypothesis that mPL-I, mPL-II, and GH may affect hepatic expression of the GHR/GHBP gene during pregnancy in the mouse.