Contribution to the study of prolactin signaling in leukocytes
Tez Türü: Doktora
Tezin Yürütüldüğü Kurum: Vrije Universiteit Brussel, Tıp Fakültesi, Belçika
Tez Danışmanı: Elisabeth Hooghe-Peters
Tezin Onay Tarihi: 2001
Tezin Dili: İngilizce
Desteklendiği Program: Diğer
Özet:
Prolactin (PRL) is a protein hormone that is synthesized and secreted primarily from the anterior pituitary gland. Also, cells of the immune system can synthesize PRL. PRL-receptors (PRL-Rs) are expressed on the surface of leukocytes. The PRL-R belongs to the hemopoietin/cytokine receptor superfamily, which activates the JAK/Stat and MAPK pathways. The wide use of the JAK/Stat pathway by many cytokines indicates the importance of this pathway in hemopoiesis and in the regulation of hemopoietic and lymphoid cell function. To evaluate the possible involvement of PRL in immune regulation, it was important to clarify by which pathways PRL acts and to identify which genes were subsequently regulated. In the present study, we have thus analyzed the signaling pathways used by PRL and monitored some effects of PRL in normal rat and human leukocytes. By RT-PCR analysis, we demonstrated that the PRL-R mRNA is expressed in rat spleen, bone marrow (BM) and thymus cells with higher expression levels in spleen and thymus than in BM. Biosynthesis of PRL-R was examined with 35S-methionine labeling followed by immunoprecipitation, SDS-PAGE and autoradiography in rat BM and spleen cells. Biosynthesis paralleled RNA expression in spleen cells. However, a discrepancy was observed in BM cells between RT-PCR products and biosynthesis (low) on one hand and Western blotting and immunocytochemistry (high) on the other hand. This discrepancy could be explained by the fact that these cells have lost part of their ability to synthesize new PRL-R but still bear functional receptors synthesized earlier. PRL promoted JAK2 tyrosine phosphorylation in rat BM and spleen cells and activated Stat-5 factor to bind gamma interferon activated sequences (GAS) from the interferon regulatory factor-1 (IRF-1) promoter in BM cells. This binding activity is correlated with the activation of the IRF-1 gene in response to PRL stimulation in both cell types. A physiological concentration of PRL was sufficient to activate the JAK/Stat signaling pathway and this activation led to the expression of the IRF-1 gene. We showed different effects of PRL in human peripheral blood mononuclear cells (PBMC) and granulocytes. In PBMC, JAK2 was constitutively phosphorylated and, after 20 min of PRL treatment (100 ng/ml), an increase in tyrosine phosphorylation was detected. In granulocytes, no phosphorylation of JAK2 could be detected after stimulation with PRL. However, tyrosine phosphorylation of JAK2 was observed in granulocyte/macrophage-colony stimulating factor (GM-CSF) treated granulocytes. After a 30 min treatment with 10 ng/ml PRL, Stat-5 was phosphorylated in PBMC but not in granulocytes. Phosphorylation of Stat-5, however, was induced in granulocytes by treatment with GM-CSF. In contrast, no phosphorylation of Stat-1 could be detected in PBMC upon PRL stimulation though IFN- induced Stat-1 phosphorylation. It is known that not all Stat activation depends on JAK. Receptor tyrosine kinases and some nonreceptor tyrosine kinases such as Src and Fyn may also phosphorylate Stat. PRL also caused a significant increase in the phosphorylation level of the p38 mitogen-activated protein kinase (MAPK) in PBMC and granulocytes. Gene expression has been studied in human PBMC, granulocytes, BM and tonsillar cells. In PBMC, suppressor of cytokine signaling (SOCS)-3 and inducible nitric oxide synthase (iNOS) gene expression levels were significantly enhanced upon 10 ng/ml PRL treatment for 30 min. In granulocytes, this physiological concentration of PRL increased IRF-1 and SOCS-2 expression and induced CIS and iNOS gene expression. In BM cells, CIS and SOCS-2 were induced by PRL. Taken together, our studies indicate that functional PRL-Rs are expressed in normal leukocytes. PRL activates JAK/Stat and MAPK signaling pathways. The induction of IRF-1 gene expression by PRL suggests an immunomodulating role for PRL. Different members of the SOCS family of proteins may modulate PRL-R signaling and PRL could either synergize with several cytokines or antagonize the effects of cytokines by inducing the expression of SOCS factors. Manipulation of PRL levels may thus have therapeutic value in some hematological disorders.