Prolactin activates mammalian target-of-rapamycin through phosphatidylinositol 3-kinase and stimulates phosphorylation of p70S6K and 4E-binding protein-1 in lymphoma cells

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac–4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.

Transgenic prolactin−/− mice: effect of trauma-hemorrhage on splenocyte functions

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL+/+ and transgenic (PRL−/−) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL−/− mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL+/+ mice. Splenocyte proliferation in male PRL−/− mice was significantly lower than in PRL+/+ mice after T-H. Marginal differences between PRL+/+ and PRL−/− mice were observed in the release of IL-2 and IFN-γ by splenocytes, while the release of IL-10 was significantly higher in PRL−/− than in PRL+/+ mice. A significant observation of our study is the release of a ∼25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL+/+ and PRL−/− mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL−/− transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice.

Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling

A prolactin (PRL)-responsive 3'-end cDNA encoding rat alpha4 phosphoprotein was previously isolated from a rat lymphoma cDNA library. Rat alpha4 is a homologue of yeast Tap42 and is a component of the mammalian target-of-rapamycin (mTOR) signalling pathway that stimulates translation initiation and G1 progression in response to nutrients and growth factors. In the present study, the full-length rat alpha4 cDNA was obtained by 5'-RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39.1 kDa. The alpha4 mRNA was expressed in quiescent PRL-dependent Nb2 lymphoma cells deprived of PRL for up to 72 h but expression was downregulated within 4 h of PRL treatment. In contrast, PRL-independent Nb2-Sp cells showed constitutive expression of alpha4 that was not affected by PRL. Western analysis of Nb2 cell lysates or of V5-tagged-alpha4 expressed in COS-1 cells detected a single immunoreactive band of approximately 45 kDa. Enzymatic deglycosylation of affinity-purified 45 kDa alpha4 yielded the predicted 39 kDa protein. Phosphorylation of Nb2 alpha4 was induced by PRL or 2-O-tetradecanoyl-phorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA. The Nb2 alpha4 associated with the catalytic subunit of protein phosphatase 2A and localized predominantly in Nb2 nuclear fractions with trace amounts in the cytosol. The immunosuppressant drug rapamycin inhibited proliferation of Nb2 cells in response to PRL or interleukin-2, but had no effect on Nb2-Sp cells. Furthermore, transient overexpression of alpha4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity. Therefore, PRL downregulation of alpha4 expression and/or PRL-inducible phosphorylation of alpha4 may be necessary for PRL receptor (PRLr) signalling to the interferon regulatory factor-1 promoter in the Nb2 cells and, furthermore, implicates cross-talk between the mTOR and PRLr signalling cascades during Nb2 cell mitogenesis.

Possible role for protein kinase B in the anti-apoptotic effect of prolactin in rat Nb2 lymphoma cells

Prolactin (PRL) is a mitogen for a number of cell types and its action as a survival factor has recently been demonstrated in Nb2 lymphoma cells. However, the intracellular signalling pathways by which PRL promotes the survival of Nb2 cells is unknown. In previous studies, we have shown that PRL caused the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its association with tyrosine phosphorylated fyn. Protein kinase B (PKB), a serine/threonine kinase, is now known to be a downstream component of the PI3-kinase pathway. The aim of the present study was to examine the effect of PRL on the activation of PKB and to find out whether this has any role on the PRL-induced survival of Nb2 cells. Our studies have revealed the phosphorylation and activation of PKB in PRL-stimulated Nb2 cells. We have also observed, using confocal microscopy, translocation of PKB to the membrane of Nb2 cells in response to PRL. These effects were blocked by the PI3-kinase inhibitor, LY294002 (10 microgram/ml). Apoptosis was induced by the general protein kinase inhibitor, staurosporine (STS; 0.1-1 microM), the synthetic glucocorticoid, dexamethasone (Dex; 100 nM) or ionising radiation by exposing Nb2 cells to X-irradiation (IR; 10 Gy). PRL had no effect on STS-induced apoptosis. On the other hand, PRL (100 ng/ml) inhibited apoptosis induced by Dex or IR; this effect of PRL was reversed by the addition of LY294002 (10 microgram/ml). Furthermore, Western blot analysis using phosphospecific PKB antibody on lysates from PRL-treated Nb2 cells showed that phosphorylation of PKB in response to PRL was inhibited by STS (0.5 microM), but not by Dex (100 nM). These results suggest that the PI3-kinase/PKB pathway may mediate the anti-apoptotic effect of PRL in Nb2 cells.

Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen

Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta. The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector. E. coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside. The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa. Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL. Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected. The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.

Changes in numbers of prolactin receptors during the cell cycle of Nb2 cells

Lactogenic hormones including prolactin (PRL) have mitogenic effects on Nb2 cells, a pre-T lymphoma cell line. Previous studies have characterized the PRL stimulation of cellular processes such as RNA/DNA synthesis, signalling molecule activation, and the expression of specific genes. The data presented here explores the fluctuations in plasma membrane PRL receptor (PRLR) number that occur in the Nb2 cells during the course of a 24 h cell cycle. PRLR abundance was determined by measuring specific binding of [I125] oPRL to G1 arrested-intact Nb2 cells in which the cell cycle was initiated by addition of nonradioactive oPRL. Preliminary studies revealed that 1 ng/ml oPRL was the minimum PRL concentration that causes a maximal stimulation of mitogenesis, without interfering with [I125] oPRL binding measurements. Subsequent experiments revealed that upon cell cycle initiation of G1 arrested Nb2 cells with 1 ng/ml oPRL, PRLR number remained constant for the initial 6 h. After 8 h PRLR numbers decreased and at 12 h, the PRLR number was less than 25% of the initial value. After 12 hr, PRLR numbers increased and reach initial values by 18 hr. These studies show that the expression of cell surface PRL receptors is modulated in a sequential fashion during the cell cycle of Nb2 cells.

Expression of prolactin mRNA and of prolactin-like proteins in endothelial cells: evidence for autocrine effects

Formation of new capillary blood vessels, termed angiogenesis, is essential for the growth and development of tissues and underlies a variety of diseases including tumor growth. Members of the prolactin hormonal family bind to endothelial cell receptors and have direct effects on cell proliferation, migration and tube formation. Because many angiogenic and antiangiogenic factors are produced by endothelial cells, we investigated whether endothelial cells expressed the prolactin gene. Here we show that bovine brain capillary endothelial cells (BBCEC) in culture express the full-length prolactin messenger RNA, in addition to a novel prolactin transcript, lacking the third exon of the gene. In addition cultures of BBCEC synthesize and secrete prolactin-like immunoreactive proteins with apparent molecular masses of 23, 21 and 14 kDa. The prolactin-like nature of these proteins in supported by the observation that Nb2-cells, a prolactin-responsive cell line, were stimulated to proliferate when co-cultured with endothelial cells and this stimulation was neutralized with prolactin-directed antibodies. Finally, consistent with a possible autocrine effect of endothelial-derived prolactins, polyclonal and monoclonal prolactin antibodies specifically inhibited basal and basis fibroblast growth-factor-stimulated growth of endothelial cells. Taken together, the present findings support the hypothesis of the prolactin gene being expressed in endothelial cells as proteins that could act in an autocrine fashion to regulate cell proliferation.

Suppression subtractive hybridization (SSH) identifies prolactin stimulation of p38 MAP kinase gene expression in Nb2 T lymphoma cells: molecular cloning of rat p38 MAP kinase

Suppression Subtractive Hybridization (SSH) has been used to compare rat Nb2 cells treated with prolactin for 1 hour with untreated cells. This new method for identifying differentially expressed genes showed that the mRNAs for at least three genes were elevated by such treatment, including a p38 mitogen activated protein (MAP) kinase. The p38 MAP kinase was cloned and the full length cDNA sequence was determined.

Prolactin induced tyrosine phosphorylation of p59fyn may mediate phosphatidylinositol 3-kinase activation in Nb2 cells

Our previous studies indicated that PI3-kinase is involved in prolactin (PRL) signalling. We have now examined the involvement of the src tyrosine kinase, fyn, in PRL-induced the activation of PI3-kinase in the rat lymphoma cell line, Nb2. Cells were stimulated with increasing doses of PRL, lysed and immunoprecipitated with anti-fyn specific antibody. Then PI3-kinase activity was measured as the increase in the phosphorylation of phosphatidylinositol to phosphatidylinositol 3-phosphate separated by TLC. Our data indicated that, in PRL treated cells, co-precipitation of PI3-kinase with anti-fyn antiserum led to time and dose-dependent activation of PI3-kinase in vitro and that this activation was blocked by the addition of LY294002. However, LY294002 appeared to have no effect on fyn autophosphorylation. Furthermore, the physical association of PI3-kinase with fyn was confirmed by Western blot analysis employing the same specific antisera. These data provide evidence that PRL-induced activation of PI3-kinase may be mediated by the tyrosine phosphorylation of fyn in Nb2 cells.