Prolactin induction of the alpha 2-Macroglobulin gene in rat ovarian granulosa cells: stat 5 activation and binding to the interleukin-6 response element.

alpha 2-Macroglobulin (alpha 2M) is expressed at high levels in the corpus luteum of pregnant rats in response to PRL and rat placental lactogens. These studies document that PRL induction of alpha 2M mRNA occurs rapidly in granulosa cells differentiated to the preovulatory phenotype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated by PRL, transient transfections of granulosa cells and electrophoretic mobility shift assays were done using the IL-6 response element (IL-6RE) of the alpha 2M promoter. The IL-6RE consists of two gamma-activating like sequences (GAS) that bind the acute phase response factor (APRF/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase reporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2M promoter can confer PRL inducibility to the luciferase reporter gene. These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts prepared from differentiated granulosa cells exposed to PRL for 0.25, 0.5, 4, and 20 h. PRL rapidly stimulated the binding of a specific protein to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assays. Because both Stat 5 and Stat 3 proteins are present in the whole cell extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial induction of the alpha 2M gene by PRL in differentiated rat granulosa cells involves, at least in part, the activation (tyrosine phosphorylation?) of Stat 5.

Regulation of mammary gland factor/Stat5a during mammary gland development.

The rat homolog of sheep mammary gland factor (MGF)/Stat5 has been isolated and used to study the regulation of Stat5 during mammary gland development and PRL regulation in COS cells transfected with Stat5a and the PRL receptor. Two alternatively spliced isoforms, designated Stat5a1 and Stat5a2, were identified, the latter encoding a carboxy-terminal truncated protein. A polyclonal antibody to a carboxy-terminal peptide of Stat5a1 was generated and used to measure the level of this isoform during mammary gland development and after PRL induction in COS cells transiently transfected with Stat5a and the long form of the PRL receptor. Surprisingly, Stat5a mRNA and protein were readily detected both in virgin rats and after mammary gland involution. The levels of Stat5a increased during pregnancy, were highest in late pregnancy, and then, unexpectedly, decreased during lactation, the time at which the highest levels of milk protein gene expression are observed. Electrophoretic mobility shift assays using the specific anti-Stat5a1 antisera demonstrated that Stat5a1 comprises part of the heterogeneous, PRL-inducible, protein-DNA complex associated with the beta-casein GAS site. Immunocytochemical analysis detected considerable cytoplasmic and some nuclear staining for Stat5a1 during late pregnancy and predominantly nuclear staining during early lactation. The lack of correspondence of Stat5a gene expression and beta-casein gene expression suggests that Stat5 activation may facilitate the interaction of other factors binding within composite response elements identified recently in the milk protein gene promoters that are then responsible for the stable expression of milk protein genes in terminally differentiated mammary epithelial cells.

High-velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo.

Mouse and rat liver, kidney and mammary gland explants were bombarded with high-velocity microprojectiles carrying a chloramphenicolacetyl transferase gene under different promoters (pTAT-cat, p chi-Casein-cat, p beta-Casein-cat). The expression of a CAT gene was revealed in all organ explants 24 h after transfection. The most pronounced expression was found when a TAT-CAT construction was used. In experiments in vivo rat liver was bombarded in situ with microprojectiles carrying pTAT-cat DNA. A marked activity of the CAT gene was detected 24 h after the bombardment.

Signal transducers and activators of transcription 5B potentiates v-Src-mediated transformation of NIH-3T3 cells.

Previously, we reported that whereas both signal transducers and activators of transcription (STAT) 5A and STAT5B can be activated with respect to tyrosine phosphorylation and DNA binding potential by Src kinase, only STAT5B was translocated to the nucleus, where it presumably activates unique downstream responses. To help elucidate the functional consequences of STAT5B activation by v-src, the properties of stably transfected NIH-3T3 cells containing both an intact and a dominant negative, COOH-terminal-truncated isoform of STAT5B were investigated. STAT5B enhanced the transforming potential of v-Src as reflected by both an increase in focus formation and growth in soft agar. STAT5B also enhanced v-Src-induced cell cycle progression and cell motility in NIH-3T3 cells. Furthermore, the dominant negative, COOH-terminal-truncated isoform of STAT5B was able to partially suppress v-Src-mediated cell transformation. These results support the hypothesis that STAT5B may enhance Src/Abl-induced tumorigenesis. Accordingly, the equilibrium between STAT5B and STAT5A and their naturally occurring truncated forms may therefore play a key role in the etiology of certain cancers.

Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A.

In this study, DNA binding and tyrosine phosphorylation of STAT5A and STAT5B were compared with their subcellular localization determined using indirect immunofluorescence microscopy. Following prolactin activation, both STAT5A and STAT5B were rapidly translocated into the nucleus and displayed a detergent-resistant, punctate nuclear staining pattern. Similar to prolactin induction, src activation resulted in tyrosine phosphorylation and DNA binding of both STAT5A and STAT5B. However, nuclear translocation of only STAT5B but not STAT5A was observed. This selective nuclear translocation appears to be mediated via the carboxyl-terminal sequences in STAT5B. Furthermore, overexpression of a dominant negative kinase-inactive mutant of JAK2 prevented prolactin-induced tyrosine phosphorylation and nuclear translocation of STAT5A and STAT5B but did not block src kinase activation and nuclear translocation of STAT5B. In co-transfection assays, prolactin-mediated activation but not src kinase-mediated activation of STAT5B resulted in the induction of a beta-casein promoter-driven reporter construct. These results suggest that STAT5 activation by src may occur by a mechanism distinct from that employed in cytokine activation of the JAK/STAT pathway, resulting in the selective nuclear translocation of STAT5B.