Prolactin binding to dissociated cells from rat mammary tumors and mammary gland.

Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.     

A novel cis-acting element in Her2 promoter regulated by Stat3 in mammary cancer cells

Stat3 plays important roles in the development of breast malignancies and oncogenesis. In the present study, a palindromic cis-acting element displaying repression activity in breast cancer cells expressing low level of Her2 was found in Her2 promoter. Deletion analysis showed that the novel element was located within Pal2 region spanning nucleotides -529 to -505. The sequence analysis of Pal2 region revealed a DNA sequence (TTAAGATAA) homologous to the binding site of Stat3, starting from position -529 to -521bp. By reporter assay, Pal2 was found to be regulated by constitutive activated Stat3C. A stimulatory effect both on Her2 mRNA and protein expressions was observed in MCF-7 cells stably expressing Stat3C, suggesting that Stat3 regulated Her2 expression. Using ChIP assays the binding of Stat3 to Her2 promoter was confirmed. The data obtained in this study indicate constitutive activated Stat3 regulates Her2 expression. Further investigation of differential effects of Stat3 exerting on breast cancer cells expressing Her2 at different levels will provide more insights into the roles of Stat3 in Her2 expression as well as the regulation of diverse biological activities.     

Receptor activator of NF-kappaB ligand induction via Jak2 and Stat5a in mammary epithelial cells

Prolactin (PRL) is the primary hormone that, in conjunction with local factors, leads to lobuloalveolar development during pregnancy. Recently, receptor activator of NF-kappaB ligand (RANKL) has been identified as one of the effector molecules essential for lobuloalveolar development. The molecular mechanisms by which PRL may induce RANKL expression have not been carefully examined. Here we report that RANKL expression in the mammary gland is developmentally regulated and dependent on PRL and progesterone, whereas its receptor RANK (receptor activator of NF-kappaB) and decoy receptor osteoprotegerin (OPG) are constitutively expressed at all stages in both normal (PRL+/-) and prolactin knockout (PRL-/-) mice. In vitro, PRL markedly increased RANKL expression in primary mammary epithelial cells and RANKL-luciferase reporter activity in CHOD6 cells, which constitutively express the PRL receptor. We identified a gamma-interferon activation sequence (GAS) in the region between residues -965 to -725 of the RANKL promoter, which conferred a PRL response. Using dominant negative mutants of recombinant Jak2 and Stat5 in CHOD6 cells, and by reconstituting the Jak2/Stat5 pathway in COS7 cells, we determined that Jak2 and Stat5a are essential for the PRL-induced RANKL expression in mammary gland.     

De-phosphorylation of GR at Ser203 in nuclei associates with GR nuclear translocation and GLUT5 gene expression in Caco-2 cells

Glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) inactivation are considered to be important in small-intestinal differentiation/maturation. In this study, we found that co-treatment with glucocorticoid hormone agonist dexamethasone and p44/42 MAPK inhibitor PD98059 in intestinal cell line Caco-2 strongly induced GLUT5 gene expression. Glucocorticoid hormone receptor (GR) was translocated from the cytoplasm to the nucleus and de-phosphorylated at serine residue 203 in the nucleus, by combined treatment with dexamethasone and PD98059. The binding of GR, as well as acetylated histones H3 and H4, to the promoter/enhancer region of GLUT5 gene was enhanced by combined treatment with dexamethasone and PD98059. These results suggest that the inactivation of p44/42 MAP kinase enhances glucocorticoid hormone-induced GLUT5 gene expression, probably through controlling the phosphorylation at serine 203 and nuclear transport of GR, as well as histone acetylation on the promoter/enhancer region of GLUT5 gene.