Prolactin (PRL) receptor signalling is essential for mammary gland function and for maintenance of the corpus luteum throughout pregnancy. PRL and other cytokines have also been shown to play important roles in breast cancer promotion. This thesis investigates the role played by a new family of signalling inhibitors, the suppressors of cytokine signalling (SOCS), in these important PRL regulated processes. SOCS were first studied as rapidly induced feedback inhibitors of immune cytokine action. Given that PRL is a class 1 cytokine and signals through the same pathways as the immune cytokines, it was anticipated that SOCS would be involved in regulating PRL signalling.
I report here (chapter 3) the cellular localisation of SOCS-3 and CIS (Cytokine- Inducible Src Homology 2-containing protein) proteins in ovary, mammary and adrenal glands of lactating rats. The expression of SOCS mRNA and protein in both ovary and adrenal gland was increased by PRL administration to lactating rat which had been deprived of circulating PRL and pups for a period of 24h. However, SOCS expression was not altered in the mammary gland of these animals in response to PRL. Moreover, suckling increased SOCS mRNA in the ovary, but not in the mammary gland, of pup-deprived rats. This differential responsiveness was associated with elevated levels SOCS-3 in the mammary gland, but not in the ovary or adrenal gland of these animals. However, deprivation of PRL and pups for a further 24h allowed the mammary gland to induce SOCS genes in response to PRL administration, and this was accompanied by a concomitant fall in basal SOCS-3 mRNA and protein expression to the level seen in other tissues. This suggests SOCS-3 induced refractoriness related to filling of the gland.
It is demonstrated here that SOCS-1, -3 and CIS, but not SOCS-2 were able to inhibit transactivation of the β-lactoglobulin promoter by PRL in transiently transfected CHO cells. In vivo, suckling resulted in loss of ovarian and adrenal responsiveness to PRL administered 2h after commencement of suckling. Taken together, it is proposed that PRL induced SOCS-1, SOCS-3 and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges during lactation, and that following pup withdrawal the milk distended mammary gland, but not the corpus luteum or adrenal cortex, is rendered unresponsive to PRL by elevated levels of SOCS-3 in manunary gland.
Prolactin and placental lactogen (PL) play key roles in maintaining the corpus luteum throughout pregnancy through activation of the PRL receptor. In chapter 4,1 have addressed the issue of whether luteolysis induced by prostaglandin F2α (PGF2α) might induce SOCS protein expression to inhibit prolactin receptor support of the corpus luteum of pregnancy. In day 19 pregnant rats, cloprostenol, a PGF2α analogue, rapidly induced transcripts for SOCS-3 and to a lesser extent, SOCS-1 in the ovary. SOCS-3 protein expression was also increased. SOCS-3 immunoreactivity was localised in the corpus luteum by immunohistochemistry. Increased SOCS-3 transcript expression was preceded by an increase in STAT3 activation, evident by 10 min after cloprostenol injection and maintained for at least 4h, with strong nuclear localisation of STAT3 visible at 2h after cloprostenol. Induction of SOCS-3 was, however, accompanied by a sharp decrease in active STAT5, and by a loss of nuclear localised STAT5. Four hours after cloprostenol administration, the corpus luteum was refractory to stimulation of STAT5 by PRL administration, and this was not due to downregulation of PRL receptor. Therefore, induction of SOCS-3 by PGF2α may be an important element in the initiation of luteolysis, acting through rapid suppression of luteotropic support from PRL.
Cytokines exert an important influence on breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Chapter 5 examines the expression of SOCS genes in a range of breast cancer lines and breast carcinomas. CIS transcript expression was elevated in all 10 breast cancer lines, although not in control lines. There was no evidence for consistent elevation of other SOCS transcripts relative to control lines. CIS protein was shown to be present in all of these lines at substantially increased levels, mainly as the 47kDa ubiquitinylated form. Expression of CIS protein did not correlate with expression of GH receptor transcripts, although in all but one case it was accompanied by elevated PRL receptor expression.
Elevated expression of SOCS-3 and CIS immunoreactive proteins were shown within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased SOCS gene expression was also observed by quantitative in situ hybridisation. Expression of the SOCS 1- 3 and CIS transcripts was seen in similar locations. A higher level of SOCS gene expression was observed within the stromal reaction of the preinvasive area relative to adjacent normal connective tissue.
The in vivo elevation of SOCS expression may be part of the host tumor response and/or it may be a response to autocrine/paracrine GH and PRL. It is proposed that the increased CIS expression in cancer lines could simultaneously shut down STAT5 signalling by trophic hormones, confer resistance to host cytokines and may increase proliferative signaling through MAP kinase pathways, since CIS is able to activate this pathway.
In conclusion, this thesis presents data supporting involvement of SOCS proteins in the regulation of PRL signalling in a tissue specific manner in order to facilitate physiological and pathological processes dependent on PRL action. These observations provide potential avenues for therapeutic and livestock applications in relation to breast cancers, lactation and the regulation of fertility.