Mechanisms underlying the diminished sensitivity to prolactin negative feedback during lactation: Reduced STAT5 signaling and up-regulation of cytokine-inducible SH2 domain-containing protein (CIS) expression in tuberoinfundibular dopaminergic neurons

Hyperprolactinaemia during lactation is a consequence of the sucking stimulus and in part due to reduced prolactin (PRL) negative feedback. To date, the mechanisms involved in this diminished sensitivity to PRL feedback are unknown but may involve changes in PRL signal transduction within tuberoinfundibular dopaminergic (TIDA) neurons. Therefore, we investigated signal transducers and activators of transcription (STAT) 5 signaling in the TIDA neurons of lactating rats. Dual-label confocal immunofluorescence studies were used to determine the intracellular distribution of STAT5 within TIDA neurons in the dorsomedial arcuate nucleus. In lactating rats with pups removed for 16 h, injection of ovine PRL significantly (P < 0.05) increased the STAT5 nuclear/cytoplasmic ratio compared with vehicle-treated mothers. In contrast, ovine PRL injection did not increase the STAT5 nuclear/cytoplasmic ratio in lactating mothers with pups, demonstrating that PRL signal transduction through STAT5 is reduced in TIDA neurons in the presence of pups. To investigate possible mechanisms involved in reduced PRL signaling, we examined the expression of suppressors of cytokine signaling (SOCS) proteins. Northern analysis on whole hypothalamus showed that CIS (cytokine-inducible SH2 domain-containing protein), but not SOCS1 or SOCS3, mRNA expression was significantly (P < 0.01) up-regulated in suckled lactating rats. Semiquantitative RT-PCR on arcuate nucleus micropunches also showed up-regulation of CIS transcripts. Immunofluorescence studies demonstrated that CIS is expressed in all TIDA neurons in the dorsomedial arcuate nucleus, and the intensity of CIS staining in these neurons is significantly (P < 0.05) increased in lactating rats with sucking pups. Together, these results support the hypothesis that loss of sensitivity to PRL-negative feedback during lactation is a result of increased CIS expression in TIDA neurons

Maximal expression of suppressors of cytokine signalling in the rat ovary occurs in late pregnancy

Maintenance of the rodent corpus luteum (CL) during pregnancy requires prolactin receptor (PRLR) signal transduction via STAT5. At the end of pregnancy, prostaglandin F2 alpha (PGF(2 alpha)) induces luteal regression through many mechanisms, including downregulation of PRLR signaling. We have previously shown that a PGF(2 alpha) analog upregulates suppressors of cytokine signaling (SOCS) proteins in the CL of day 19 pregnant rats leading to reduced STAT5 signaling. Here, we examined endogenous SOCS expression and STAT5 signaling in the rat ovary during normal pregnancy and luteolysis. The mRNA expression of Socs1, Socs2, and Socs3 and related cytokine-inducible SH2-containing protein (Cish) was low in early pregnancy (day 7), but significantly increased at mid-pregnancy (days 10 and 13) associated with increased endogenous tyrosine phosphorylation (TyrP) of STAT5. In support of the notion that these changes are due to increasing placental lactogen levels at this time, we found that treatment with exogenous PRL on day 7 increased TyrP of STAT5 and induced SOCS mRNA expression, except Socs3. After mid-pregnancy, further significant increases in Socs3 and Cish mRNA expression were observed. Such changes in mRNA expression correlated with protein levels, with protein levels of both SOCS3 and CISH being maximal in late pregnancy (days 19-21). In addition, a significant reduction in TyrP of STAT5 was first observed on day 20, with a further substantial decrease on day 21. Therefore, these results are consistent with the hypothesis that increased SOCS expression in the rat ovary during late pregnancy reduces STAT5 signaling, which may be important in PGF(2 alpha)-induced luteolysis. Reproduction (2009) 138 537-54

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