STAT5 expression and regulation in mammary epithelial cells

Sammanfattning: The mammary gland of an adult mammal undergoes cycles of proliferation and differentiation influenced by growth factors, peptide, and steroid hormones, e.g. epidermal growth factor (EGF), prolactin (PRL) and estrogen (E). The mouse mammary epithelial cell line HC 11 has proven to be a valuable tool for investigation of molecular mechanisms taking part in the processes of development of the mammary gland. PRL signaling through the PRL receptor and the receptor-associated Janus kinase 2 (JAK2) can activate latent cytoplasmic transcription factors of the signal transducer and activator of transcription (STAT) family of proteins. In particular the two STAT5 isoforms, STAT5A and STAT5B, are associated with effects of PRL. In this thesis expression and activity of the JAK-STAT pathway in HC11 cells was investigated with focus on STAT5A. We found that STAT5 was expressed in HC11 cells during proliferation and differentiation. A marked increase in STAT5 expression was seen at the onset of differentiation. Our data suggest that EGF repression of STAT5 expression through the ras/raf/MEK/MAPK and PI-3 kinase pathway is responsible for the lower expression during proliferation. The 5' flanking region of the mouse STAT5A gene was isolated and four putative peroxisome proliferator-response elements (PPRE) were found. Treatment of HC11 cells with PPARgamma ligand increased the expression of endogenous STAT5A and of transiently transfected reporter gene constructs harbouring 3.9 kb of the mouse STAT5 5'-flanking region. Transient transfection of constructs containing 5' to 3' deletion or mutated PPRE indicated that at least three PPREs are involved in the regulation of the STAT5A gene. Activity of an estrogen response element in a reporter gene construct (ERE-reporter) and of the betacasein promoter fused to a reporter gene was analyzed in HC11 cells. E-induced EREreporter gene expression was found only in proliferating cells and was dependent on EGF. Our data indicated that this could be due to EGF-repression of corepressors. PRL-induced beta-caseinreporter expression was only detected in differentiated cells. This PRL-induced reporter expression was decreased by E. ER was found to interact with STAT5, which could explain the repressive effect of E on beta-casein promoter activity. Hyperactive JAK2 mutants were stably expressed in HC11 cells. One mutant, consisting of the kinase domain, induced increased nuclear translocation of STAT5, which did not correlate with DNA-binding activity. Decreased ability to undergo apoptosis in HC11 cells expressing JAK2 mutants implicated that hyperactive JAK has a transforming potential.