Prolactin signaling and Stat5: going their own separate ways?

Miyoshi et al. compared the role of the prolactin receptor (PrlR) and its downstream mediator, the signal transducer and activator of transcription 5 (Stat5), in mammary epithelial cells in vivo by studying PrlR(-/-) and Stat5ab(-/-) mouse mammary epithelial transplants during pregnancy. At first glance, the two mutant epithelia appear to have similar defects in the differentiation of the alveolar epithelium. However, a closer examination by Miyoshi et al. revealed defects in the epithelial architecture of the smallest ducts of Stat5ab(-/-) transplants not apparent in the PrlR(-/-) transplants, suggesting that Stat5 is more than a simple mediator of PrlR action

Characterization of the SOCS3 Promoter Response to Prostaglandin E2 in T47D Cells

Suppressor of cytokine signaling 3 ( SOCS3), a negative regulator of cytokine signaling, is expressed in breast cancer cells where it can modify sensitivity and responsiveness to cytokine signaling through the Janus kinase/ signal transducer and activator of transcription ( JAK/ STAT) pathways. Although it is widely accepted that SOCS3 expression is in itself regulated by STATs, we and others have shown that prostaglandins can also up- regulate SOCS3 expression. Here we used T47D breast cancer cells treated with prostaglandin E-2 ( PGE(2)) to examine this pathway. T47D cells responded to PGE(2) stimulation with a significant increase in SOCS3 mRNA that was independent of de novo protein synthesis. PGE(2) stimulation resulted in STAT3 serine and tyrosine phosphorylation, although mutation of either of the two previously characterized STAT response elements on the SOCS3 promoter did not affect SOCS3 promoter activation by PGE(2). In addition, overexpression of STAT3 wild- type, constitutively active or dominant-negative constructs did not affect PGE(2) induced SOCS3 promoter activation, indicating that STATs are unlikely mediators of this pathway in these cells. PGE(2) is a known activator of the cAMP/ protein kinase A ( PKA) pathway, and in T47D cells, up- regulation of SOCS3 mRNA by PGE(2) was abolished by pretreatment with H89, a PKA inhibitor and increased by cAMP and forskolin treatment. Consistent with this, PGE(2) treatment increased cAMP response element ( CRE)- binding protein serine phosphorylation. However, mutation of the activator protein 1/ CRE on the promoter did not affect basal or PGE(2)- stimulated activation, suggesting a role for cAMP/ PKA that is independent of CRE- binding protein binding. Mutation of the GC- rich region of the SOCS3 promoter, a putative Sp1/ Sp3 binding site, abolished both basal and PGE(2)- stimulated activation. Gel- shift assays showed increased complex formation after treatment, and this was inhibited by the addition of an Sp1 antibody or pretreatment with PKA inhibitor. Chromatin immunoprecipitation assay verified Sp1 binding to the promoter in response to PGE(2). Sp1 overexpression increased SOCS3 promoter activation, and both basal and PGE(2)- induced SOCS3 mRNA expression was prevented by mithramycin, an inhibitor of Sp1 DNA binding. Finally, a physiological role for PGE(2) was demonstrated with PGE(2) pretreatment reducing lipopolysaccharide- induced STAT3 activation. Collectively, this study details a novel mechanism of SOCS3 up- regulation by PGE(2) in breast cancer cells that appears to be STAT independent and involve Sp1 binding to the promoter. This process has possible implications for cytokine responsiveness and tumor progression