Progestins induce transcriptional activation of Signal Transducer and

ELIZALDE PV ; PROIETTI CJ ; SALATINO M ; SCHILLACI R ; CARNEVALE R ; ROSEMBLIT C ; PECCI A ; KORNBLIHTT A ; CHARREAU EH

Orlando

Congreso; AACR Annual Meeting; 2004

American Associatiom of Cancer Research (AACR)

Interactions between steroid hormone receptors and signal transducers and activators of transcription (Stats)-mediated signaling pathways have been found. This cross-talk has a bi-directional nature, where steroid hormone receptors regulate Stat-dependent transcription and conversely, Stats are able to modulate steroid hormone-mediated transcription. Here, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice. We found that primary cultures of C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 at protein level and that MPA treatment of C4HD cells for 48-h up-regulated Stat3 protein expression. Furthermore, MPA was able to induce Stat3 tyrosine phosphorylation after 5-10 min stimulation. Subcellular fractionation studies showed that MPA treatment of C4HD cells significantly increased nuclear localization of Stat3. MPA also induced Stat3 association with the progesterone receptor (PR), as evidenced by coimmunoprecipitation experiments in C4HD cells. Electrophoretic mobility shift assays (EMSA) showed that MPA treatment of C4HD cells for 15 min induced Stat3 binding to DNA, using as labeled probe either the sis-inducible element (SIE) of the human c-fos promoter or the interleukin-6-inducible high affinity Stat3-responsive site within the rat alpha(2)-macroglobulin promoter. Presence of specific Stat3-DNA complexes after MPA treatment was demonstrated by competition with excess unlabeled oligonucleotides, and by lack of competition with mutated oligodeoxinucleotide probes. The inclusion of a rabbit polyclonal anti-Stat3 antibody in the EMSA reaction supershifted the protein-DNA complex. An equivalent amount of preimmune rabbit serum, used as control, had no effect. These data clearly show the presence of Stat3 in the DNA-protein complex. MPA-induced Stat3 binding to DNA is mediated by the classic PR, as indicated by the finding that the progestin antagonist RU486 completely abolished MPA effect. Transient transfections of C4HD cells with a luciferase reporter gene containing four copies of the m67 high-affinity Stat 3 binding site gene, demonstrated that MPA promoted a significant increase in luciferase activity, which was inhibited by RU486. These results provide the first evidence that progestins, acting through the classic PR, are able to induce transcriptional activation of Stat3 in mammary tumor cells.