IRF-1 AND PROLACTIN IN T CELL ACTIVATION

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The peptide hormone prolactin (PRL) exerts a profound effect on both cellular proliferation and differentiation in a variety of tissues, but its role as an immunomodulator has not been well-defined. That PRL plays a potentially important role as a novel T and B cell cytokine has been strengthened by the observations that the PRL receptor (PRL-R), a member of the cytokine receptor superfamily, is expressed on cells of the immune system, and that some of these cells also synthesize and secrete biologically-active PRL. The long-term objective of this proposal is to understand the immunoregulatory properties of PRL. As a model system for investigating the role of PRL as a T cell cytokine, we are using the rat Nb2 T lymphoma cells which require PRL for growth. A major target of PRL stimulation in Nb2 T cells is the transcription factor, interferon regulatory factor-1 (IRF-1). In T cells, PRL stimulates the biphasic expression of IRF-1, first during G1 activation, and again over G1/S transition, where its expression is tightly linked to DNA synthesis and subsequent cell proliferation. Our recent studies have implicated the cytokine signaling molecules "signal transducer and activator of transcription" or Stat, and the cell cycle-regulated transcription complex containing retinoblastoma protein (Rb) as possible PRL signaling molecules at the IRF-i promoter. Our working hypothesis is that PRL and its receptors are involved in signaling for G1 activation as well as for S phase progression in activated T cells. Both cytokine and cell cycle signaling molecules are activated at the IRF-I promoter in a biphasic manner. We further suggest that the transcription factor IRF-1 is a nuclear mediator of PRL action during these distinct phases of the T cell cycle. The proposed studies aim to elucidate how cytokine signal transduction may be integrated with cell cycle control signals to regulate IRF-1 expression, and how IRF-1 may be important for T cell activation and proliferation. Studies are proposed to: 1) Characterize the biphasic PRL response at the IRF-1 promoter during G1 versus S phase, and to elucidate how Stat and Rb proteins participate in these responses. Mutational analysis of promoter elements coupled with studies of protein!DNA interactions will help to identify cis-acting elements and the transacting factors important for PRL stimulation of the IRF-1 gene; and 2) Investigate IRF-1 function and PRL action in T cells. CD8+ T cells from thymocytes and splenocytes representing different stages of development will be analyzed as potential targets of PRL immunomodulation. IRF-1 target genes will also be cloned by differential display PCR, as one way of understanding how PRL (a cytokine signal) and IRF-1 (one cytokine target) modulate T cell activation and proliferation. These studies represent a comprehensive molecular, genetic, biochemical and immunological approach to defining how cytokine signals are integrated with cell cycle signals to regulate IRF-1 gene expression in T lymphocytes. Understanding how PRL modulates the biological functions of T cells should elucidate the immunoregulatory properties of PRL, and provide new insights into neuroendocrine-immune system interactions.