Prolactin-dependent activation of a tyrosine phosphorylated DNA binding factor in mouse mammary epithelial cells.

Abstract

The mammary gland factor MGF has been described as a developmentally and environmentally regulated nuclear factor required for transcription of the milk protein gene beta-casein. In the current study the individual role of lactogenic hormones in the activation of MGF DNA binding and the functional relation of MGF to known transcription factors was investigated by electrophoretic mobility shift assays. DNA binding of MGF was rapidly induced by PRL in mammary epithelial cells. The activation was not inhibited by the protein synthesis inhibitor cycloheximide. The effect of PRL on MGF did not require costimulation of cells with the other lactogenic hormones, insulin, and glucocorticoids. Thus, MGF is the first example of a nuclear factor directly regulated by PRL. The MGF complexes formed upon initiation of lactation in the mammary gland and upon stimulation of mammary epithelial cells with PRL migrated at the same position in electrophoretic mobility shift assay, whereas the MGF complex found in mammary gland extracts of pregnant mice exhibited a faster mobility. In cell cultures, PRL-induced activation of MGF as well as up-regulation of beta-casein gene transcription was confined to confluent cultures of mammary epithelial cells and inhibited by long term incubation of cells with epidermal growth factor. MGF was found to be related to the nuclear factors that are activated by tyrosine phosphorylation when cells are stimulated with interferons or cytokines. This notion is supported by experimental evidence for phosphorylation of MGF on tyrosine and by the similar DNA recognition motifs of MGF and cytokine-activated factors.

Dominance of IL-12 Over IL-4 in γδ T Cell Differentiation Leads to Default Production of IFN-γ: Failure to Down-Regulate IL-12 Receptor β2-Chain Expression

“…The double-stranded 32 P-labeled oligonucleotide 5Ј-GTGCATTTCCCG TAAATCTTGTCTACAATTC-3Ј (m67) and annealed complementary oligonucleotide were used as described (28). Binding reactions with wholecell extracts and EMSA on 4% polyacrylamide gels were also performed as described previously (28).…”

“…Binding reactions with wholecell extracts and EMSA on 4% polyacrylamide gels were also performed as described previously (28).…”

Retinoic acid modulates prolactin receptor expression and prolactin-induced STAT-5 activation in breast cancer cells in vitro

“…One attractive model explaining the rapid suppression of PRL-R, even at very low doses of RA, could be the interference between the above-mentioned nuclear pathways. To investigate the functional importance of this process, we studied the modulation of a PRL-dependent intracellular signal transduction pathway by retinoids, namely the activation of STAT-5, a factor known to be activated by PRL (Welte et al, 1994). Pretreatment of T47D cells with 9-cis-RA suppresses PRLmediated STAT-5 activation, demonstrating a functional effect of RA-mediated down-regulation of PRL-R.…”

“…Assays were performed by a method similar to that described by Welte et al (1994). Oligonucleotides were purified by polyacrylamide gel electrophoresis, radioactively labelled with [γ-32 P]ATP (>6000 Ci mmol -1 ) and T4 polynucleotide kinase, and purified by phenol extraction and Sephadex G50 chromatography.…”

“…We, therefore, studied the modulation of expression of PRL-R mRNA by retinoids in the human breast cancer cell lines MCF-7, SKBR-3, T47D and BT-20 applying Northern blot and a quantitative polymerase chain reaction (PCR) method. Because one of the most important mediators of PRL-regulated genes is STAT-5 (Welte et al, 1994), we were interested in whether this transcription factor is also modulated by RA.…”