Regulation of SOCS3 by growth hormone, prolactin and prostaglandin E2

Suppressor of cytokine signalling (SOCS) 3 is a negative regulator of cytokines and growth factors that signal through class I cytokine receptors, and as such, modulates the responsiveness of cells to cytokine stimulation. One of the dominant signalling pathways employed by cytokines is the Janus kinase/Signal transducers and activators of transcription (JAK/STAT) pathway, and SOCS3 is a product of this pathway, and specifically inhibits this pathway, in a negative feed-back loop. SOCS3 signaling contributes substantially to immune function and response to immune challenge, development and function of reproductive tissues in particular the mammary gland, and energy homeostasis. Deregulation of cytokine signalling has been implicated in numerous disease states, including tumourogenesis, making SOCS3 an attractive therapeutic target for the treatment of such conditions. In order for SOCS3 expression to be manipulated for therapeutic use, it is crucial that the signalling pathways that are involved in its regulation in response to different ligands are elucidated. This thesis presents three models of SOCS3 transcriptional regulation. Chapter 2 details SOCS3 regulation in response to GH treatment in a pro-B cell line that has been stably transfected with the human growth hormone receptor, BaF3/hGHR cells. In this haematopoietic model, SOCS3 expression following GH treatment is dependent on JAK activation, and the phosphorylation of STAT5b, which binds to the proximal STAT response element (SRE) on the SOCS3 promoter. In addition, GH-induced SOCS3 expression is also dependent on Jun-activated kinases (JNK) and p38 mitogen-activated kinase (MAPK), the recruitment of c-Fos and c-Jun to the AP1/CRE motif, and FKHRL to the overlapping FOXO regulatory element on the promoter. In contrast, CREB, which is constitutively bound to the AP1/CRE element on the SOCS3 promoter, is removed following GH treatment, although remains associated with the c-Fos/c-Jun complex. Finally, overexpression of SOCS3 protein in BaF3/hGHR cells causes decreased proliferation, whilst transfection with SOCS3 siRNA causes increased proliferation, and augments GH-induced proliferation. This study presents novel findings for the regulation of SOCS3 by GH. Chapter 3 is a study of PRL-induced SOCS3 expression in T47D breast cancer cells. Similar to the GH study, SOCS3 expression in T47D cells following PRL treatment is dependent on STATs binding to the proximal SRE on the promoter. However T47D cells possess constitutively active STAT3 and STAT5, and PRL treatment causes activation of STAT1 and further activation of STAT5. Gel shift assays reveal that STAT1, 3 and 5 are all capable of binding to the SOCS3 promoter depending on cellular conditions, although it appears that STAT5 is the most potent activator of the SOCS3 promoter. In addition, PRL-induced STAT5 activation is, in part, independent of JAK activation, as is SOCS3 expression. Finally, SOCS3 promoter activity in T47D cells is dependent on the GC-rich region, and mutation of this region reduces basal promoter activation and prevents further activation by PRL. Sp1 was found to bind to this region of the promoter in a protein kinase a (PKA)-dependent manner. This is the first study to show the involvement of Sp1 on SOCS3 regulation. Chapter 4 outlines the signalling pathway employed by prostaglandin E2 (PGE2), a lipid mediator involved in immune function and implicated in tumourogenesis, in the regulation of SOCS3 expression in T47D cells. In contrast to PRL- and GH-stimulated SOCS3 expression, PGE2 treatment causes upregulation of SOCS3 expression independently of STAT activation. Rather, PGE2 signals through the G-protein coupled receptor EP4 to cause PKA-dependent recruitment of Sp1 to the GC-rich region of the SOCS3 promoter, and this alone is sufficient for the initiation of transcription. Finally, overexpression of SOCS3 in T47D cells causes a significant reduction in STAT3 expression, whilst having little effect on STAT1 or STAT5 activation or expression. STAT3 plays an important role in the mammary gland during involution, where it induces apoptosis. However in breast cancer cells STAT3 is thought to be pro-oncogenic, and its expression and activation is associated with a proliferative and metastatic phenotype. Therefore it was surprising that overexpression of SOCS3 caused increased proliferation in T47D cells, given its effects on STAT3 expression. Together these three studies provide novel findings as to the regulation of SOCS3 expression, and indicate that this process is highly dependent on the cellular environment, which is influenced by the cell type in question, and the stimulating ligand. In light of this information, SOCS3 expression could be elegantly regulated in a cell- and ligand-specific manner, with obvious therapeutic potential