Lewis G. Sheffield

Production and regulation of leptin in bovine mammary epithelial cells

Previously, leptin has been found in human and rodent mammary tissue. The present research was conducted to determine (1) if leptin is produced by bovine mammary epithelial cells and (2) if leptin production in bovine mammary epithelial cells is hormonally regulated. Western blot analysis indicated the presence of leptin in bovine milk, while reverse transcription-polymerase chain reaction (RT-PCR) indicated the presence of leptin mRNA in mammary tissue and cultured bovine mammary epithelial cells (MAC-T cell line). A real time RT-PCR method was developed that allowed quantitative assessment of bovine leptin mRNA over approximately 3 orders of magnitude. Time course studies indicated a rapid increase in leptin mRNA in response to insulin or insulin-like growth factor-I (IGF-I). When normalized against bovine GAPDH as an internal control, 0.5 or 1h treatment with 10 ng/mL insulin gave 39+/-4 and 64+/-2-fold increase in leptin mRNA compared with 0 h control. Leptin mRNA was increased 257+/-9 and 75+/-23-fold by 0.5 or 1h treatment with 10 ng/mL IGF-I. Dose response studies indicated significant increases in leptin mRNA in response to as little as 1 ng/mL insulin or 0.1 ng/mL IGF-I. Maximum increase in leptin mRNA was observed in response to 10 ng/mL insulin and 10 ng/mL IGF-I. These results indicate that production of leptin by bovine mammary epithelial cells can be regulated by factors known to alter mammary function and nutrient partitioning. This suggests that leptin may be an autocrine/paracrine signal in the bovine mammary gland.

Prolactin decreases epidermal growth factor receptor kinase activity via a phosphorylation-dependent mechanism.

Previously, we have shown that prolactin inhibits epidermal growth factor (EGF)-induced mitogenesis in mouse mammary epithelial cells without altering the response to other growth promoting agents. This effect has been associated with reduced EGF-induced EGF receptor (EGFR) tyrosine phosphorylation, Grb-2 association, and Ras activation. Our current hypothesis is that prolactin induces an alteration in EGFR kinase activity via a phosphorylation-dependent mechanism. To test this hypothesis, we treated normal murine mammary gland cells with or without 100 ng/ml prolactin. EGFR isolated by wheat germ agglutinin affinity chromatography from nontreated cells exhibited substantial ligand-induced phosphorylation, and EGFR isolated from prolactin-treated cells displayed minimal EGF-induced EGFR phosphorylation, as well as decreased kinase activity toward exogenous substrates. The observed decrease in ligand-induced EGFR phosphorylation could not be attributed to either differential amounts of EGFR, decreased EGF binding affinity, or the presence of a phosphotyrosine phosphatase or ATPase. EGFR isolated from prolactin-treated cells exhibited increased phosphorylation on threonine. Removal of this phosphorylation with alkaline phosphatase restored EGFR kinase activity to levels observed in nontreated cells. Therefore, these results suggest that prolactin antagonizes EGF signaling by increasing EGFR threonine phosphorylation and decreasing EGF-induced EGFR tyrosine phosphorylation.

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin + hydrocortisone and insulin + prolactin + hydrocortisone increased both prepro-EGF and beta-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.

Influence of Submandibular Salivary Glands on Hormone Responsiveness of Mouse Mammary Glands

Abstract Surgical removal of the submandibular salivary glands (sialoadenectomy) of female Balb/c mice significantly (P < 0.05) reduced mammary development as judged by development scores and mammae DNA levels. Reduction in mammae development score by sialoadenectomy was observed in both mice saline injected and mice treated with estradiol and progesterone. Autografts of submandibular salivary tissue or daily administration of EGF to sialoadenectomized mice partly alleviated the atrophy of the mammary gland induced by sialoadenectomy (P < 0.05). The results of our studies are consistent with a model of mammary gland developmental regulation that includes the submandibular salivary gland as a mediator of mammogenesis via secretion of EGF.

Cyclic Nucleotides and Protein Phosphorylation in Mouse Mammary Glands: Effects of Estrogen and Progesterone Administered in Vivo

Abstract Ovariectomized mice were injected daily for 20 days with saline, 17β-estradiol (1 μg/ day), progesterone (1 mg/day), or estrogen + progesterone. Mammary glands were removed, homogenized, and analyzed for DNA, cAMP, cGMP, cAMP-dependent protein kinase (kinase A), cGMP-dependent protein kinase (kinase G), tyrosyl kinase (kinase T), and epidermal growth factor-stimulated tyrosyl kinase (EGF-T). Estrogen and progesterone, administered singly, increased DNA, cAMP, kinase A, kinase T, and EGF-T. In addition, progesterone, administered alone or with estrogen, decreased kinase G activity. cGMP concentrations were not altered by estrogen or progesterone. No evidence of a synergism between estrogen and progesterone on the levels of the cyclic nucleotides and the activities of kinase enzyme was observed, although an additive effect of these steroids was seen. These data indicate that ovarian steroid-induced growth of mouse mammary glands is accompanied by significant changes in protein phosphorylation, i.e., increased cAMP-dependent protein phosphorylation and tyrosyl phosphorylation and decreased cGMP-dependent protein phosphorylation.

Adenosine Stimulation of DNA Synthesis in Mammary Epithelial Cells

Abstract Adenosine increased the DNA synthesis rate and the percentage of S-phase cells 2–3-fold in mouse mammary epithelial cells (NMuMG), with an optimum concentration of 10–100 μM. This effect was not mimicked by adenosine metabolites adenine, hypoxanthine, or inosine. N-ethylcarboxamidoadenosine (NECA, a relatively nonselective adenosine receptor agonist) and 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarbox-amidoadenosine (CGS-21680, an A2 selective agonist) also increased DNA synthesis by mammary epithelial cells. However, N6-cyclohexyladenosine (CHA, an agonist for A1 type receptors) decreased DNA synthesis. The A1 selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on basal or adenosine-induced DNA synthesis, whereas the A2 selective antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) decreased adenosine-induced DNA synthesis. Similar effects were observed in another nontumorigenic mouse mammary epithelial line, HC11, as well as the nontumorigenic human lines MCF-10A and 184.A1. Binding studies indicated that NMuMG cells contained approximately 3200 A1 receptors and about 5300 A2 receptors per cell. Both CGS-21680 and CHA increased GTPase activity in isolated cell membranes, whereas only CGS-21680 increased activity of adenylyl cyclase. Adenosine and CGS-21680 increased expression of a cyclic AMP responsive reporter gene. In addition, the protein kinase A inhibitor H-89 blocked the ability of adenosine and CGS-21680 to induce DNA synthesis, but did not affect EGF-induced DNA synthesis. These results indicate that adenosine appears to be a possible growth promoting agent in mammary tissue, and this effect may be mediated by extracellular receptors of the A2 type.

Malignant Transformation of Mammary Epithelial Cells Increases Expression of Leptin and Leptin Receptor

Both normal and malignant mammary tissues have been shown to produce leptin and express leptin receptors. This study compared the expression of leptin and leptin receptor mRNA in a variety of normal and malignant mammary epithelial cell lines and observed that in general the malignant lines expressed higher levels of leptin and leptin receptor mRNA than nonmalignant lines. Furthermore, oncogenic transformation of nonmalignant cell lines increased expression of leptin and leptin receptor, with expression of ErbB2 giving particularly high levels of expression of long-form leptin receptor. In addition, nonmalignant cells exhibited little or no increase in DNA synthesis following leptin treatment, whereas oncogene-transformed cells had increased DNA synthesis in response to leptin. These effects varied among oncogenes, with ErbB2-transformed cells showing particularly high expression of leptin receptor mRNA and high response to leptin.

OLIGONUCLEOTIDES ANTISENSE TO CATALYTIC SUBUNIT OF CYCLIC AMP-DEPENDENT PROTEIN KINASE INHIBIT MOUSE MAMMARY EPITHELIAL CELL DNA SYNTHESIS

Mouse mammary epithelial cells were plated onto 24-well culture plates (50,000 per well), allowed to attach and serum starved for 24 h. Following serum starvation, DNA synthesis was induced by the addition of 10% fetal calf serum and determined by a 1-h pulse with [3H]thymidine from 17 to 18 h after serum addition. Addition of oligonucleotides antisense to the translation start region of cyclic AMP-dependent protein kinase (kinase A) mRNA inhibited thymidine incorporation into DNA (total or percentage of cells incorporating thymidine, as measured by autoradiography). This inhibition was apparent whether compared to controls with no oligonucleotide addition, sense oligonucleotides, or mismatch oligonucleotides. Enzymatic assays indicated that the antisense oligonucleotides lowered kinase A activity in cells. Time course studies indicated that the inhibition in DNA synthesis was not an artifact of the time at which DNA synthesis was estimated. Long-term (4 day) cultures indicated that effects on induction of DNA synthesis were reflected in long-term cell proliferation.

Influence of epidermal growth factor on growth of bovine mammary tissue in athymic nude mice

Bovine mammary tissue obtained from midpregnant Holstein heifers by surgical biopsy was transplanted subcutaneously to ovariectomized athymic nude mice (n = 5 heifers). After 3 weeks recovery, mice were either sham operated or sialoadenectomized (submandibular salivary glands removed). After an additional week, mice were injected with saline or 17 beta-estradiol + progesterone (1 microgram + 1 mg/day) for 2 days. In addition, half of the sialoadenectomized mice were injected with epidermal growth factor (5 micrograms/day). Grafted tissue was removed and rate of deoxyribonucleic acid (DNA) synthesis estimated by incorporation of 3H thymidine. Estradiol + progesterone increased the incorporation of 3H thymidine from 77 +/- 20 dpm/micrograms DNA to 472 +/- 53 dpm/micrograms DNA. In sialoadenectomized mice, DNA synthesis was increased from 88 +/- 16 dpm/micrograms DNA (saline treated) to 360 +/- 29 dpm/micrograms DNA (estradiol + progesterone treated). In sialoadenectomized mice treated with epidermal growth factor, DNA synthesis in estradiol + progesterone treated mice was 529 +/- 36 dpm/micrograms DNA, compared to 112 +/- 30 dpm/micrograms DNA in sialoadenectomized mice treated with epidermal growth factor. These data indicate that sialoadenectomy of athymic nude mice decreased the ability of transplanted bovine mammary tissue to increase DNA synthesis in response to estradiol and progesterone. This inhibition was removed by epidermal growth factor treatment. These data suggest a physiological role of epidermal growth factor in regulating development and hormone responsiveness of bovine mammary tissue.

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.