Ž . Journal of Neuroimmunology 94 1999 40–47 Requirement of PI3-kinase activity for the nuclear transport of prolactin in cloned murine T lymphocytes Stanley M. Belkowski, Jason E. Levine, Michael B. Prystowsky ) Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park AÕenue, Bronx, New York, NY 10461, USA Received 10 June 1998; revised 20 August 1998; accepted 21 August 1998 Abstract The murine T-cell clone, L2, requires both IL2 and PRL to proliferate. Proliferation and selected IL2-driven gene expression are blocked by treatment with rapamycin. Since prolactin translocation to the nucleus is IL2 dependent and required for proliferation, experiments were performed to identify activation pathways that might be involved in nuclear transport and proliferation. L2 cells were Ž . stimulated with IL2 in the presence and absence of the mTOR inhibitor rapamycin, PI3-kinase inhibitors wortmannin, LY294002 , the p38 MAP kinase inhibitor SB203580 and the vitamin D analog calcipotriol. The immunosuppressant rapamycin markedly inhibited IL2-induced proliferation and prolactin translocation to the nucleus. Similarly, wortmannin and LY294002 inhibited IL2-induced proliferation and markedly decreased the amount of prolactin transported to the nucleus. SB203580 and calcipotriol partially inhibited IL2-induced proliferation but had no effect on prolactin translocation. None of the inhibitors affected Lucifer Yellow uptake indicating that rapamycin, wortmannin and LY294002 did not inhibit early endosomal formation but rather worked to inhibit prolactin translocation at a later point in the retrograde transport pathway. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Prolactin; T-cell activation; PI3-kinase; Wortmannin; Rapamycin 1. Introduction A growing body of evidence supports an immunoregu- Ž . latory role for prolactin PRL . In vivo animal studies have demonstrated that the injection of PRL induces lymphoid Ž . hyperplasia Berczi et al., 1981 . Conversely, post-hy- Ž pophysectomy induced immunosuppression Nagy and . Berczi, 1978, 1991 is largely reversible by the exogenous administration of PRL. Similarly, in vivo administration of the inhibitor of PRL secretion, bromocryptine inhibits Ž T-cell proliferation and production of cytokines Nagy et . al., 1983; Bernton et al., 1988 . Anti-PRL antisera specifi- cally inhibit the in vitro proliferation of IL2, concanavalin Ž A and lipopolysaccaharide-driven lymphocytes Hartmann . et al., 1989; Clevenger et al., 1990a , demonstrating that PRL is necessary for lymphocyte proliferation. PRL recep- tors have been identified on both B and T lymphocytes ) Corresponding author. Tel.: q1-718-4302827; Fax: q1-718-4308541 Ž Montgomery et al., 1987; Matera et al., 1988; Pellegrini et . al., 1992 , and the PRL-induced upregulation of IL2 Ž . Ž . Mukherjee et al., 1990 and transferrin Gout et al., 1988 receptors on T-cells further suggests an immunoregulatory role for PRL. In studies using a homogeneous population of cloned murine T cells, the secretion but not the synthe- Ž . sis of PRL has been shown Clevenger et al., 1990a . Performed in chemically defined medium, these studies have indicated an ability of murine T cells to sequester PRL from the external medium, with increased secretion of sequestered PRL occurring during IL2 stimulation. PRL in part mediates its effects on the immune system through the PRL receptor, which has been identified on Ž both B and T lymphocytes Montgomery et al., 1987; . Matera et al., 1988 and appears necessary for in vivo and Ž in vitro immune response Nagy and Berczi, 1978; Berczi et al., 1981; Nagy et al., 1983; Bernton et al., 1988; . Clevenger et al., 1990a . The PRL receptor is structurally related to the gene superfamily of growth hormone, ery- thropoiten, and granulocyte-macrophage colony-stimulat- Ž . ing factor Kelly et al., 1991 . PRL binding to the PRL receptor initiates kinase cascade activation which induces 0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0165-5728 98 00202-1