MAMMARY-GLAND FACTOR (MGF) IS A NOVEL MEMBER OF THE CYTOKINE REGULATED TRANSCRIPTION FACTOR GENE FAMILY AND CONFERS THE PROLACTIN RESPONSE

Milk protein gene expression in mammary epithelial cells is regulated by the action of the lactogenic hormones insulin, glucocorticoids and prolactin. The mammary gland factor, MGF, has been shown to be a centr al mediator in the lactogenic hormone response. The DNA binding activi ty of MGF is hormonally regulated and essential for beta-casein promot er activity. We have used Red A Sepharose- and sequence-specific DNA a ffinity chromatography to purify MGF from mammary gland tissue of lact ating sheep. Proteins of 84 and 92 kDa were obtained, proteolytically digested and the resulting peptides separated by reverse phase high pr essure liquid chromatography. The 84 and 92 kDa proteins yielded very similar peptide patterns. The amino acid sequence of two peptides was determined. The sequence information was used to derive oligonucleotid e probes. A cDNA library from the mRNA of mammary gland tissue of lact ating sheep was screened and a molecular clone encoding MGF was isolat ed. MGF consists of 734 amino acids and has sequence homology with the 113 (Stat113) and 91 kDa (Stat91) components of ISGF3, transcription factors which are signal transducers of IFN-alpha/beta and IFN-gamma. Two species of MGF mRNA of 6.5 and 4.5 kb were detected in mammary gla nd tissue of lactating sheep. Lower mRNA expression was found in ovary , thymus, spleen, kidney, lung, muscle and the adrenal gland. MGF cDNA was incorporated into a eukaryotic expression vector and cotransfecte d with a vector encoding the long form of the prolactin receptor into COS cells. A strong MGF-specific bandshift was obtained with nuclear e xtracts of COS cells induced with prolactin. Treatment of activated MG F with a tyrosine-specific protein phosphatase resulted in the loss of DNA binding activity. Prolactin-dependent transactivation of a beta-c asein promoter-luciferase reporter gene construct was observed in tran sfected cells.