Multiple cAMP-induced signaling cascades regulate prolactin expression in T cells.

Beside its pivotal role in reproduction, the pituitary hormone prolactin (PRL) has been attributed an immunomodulatory function. Here we report that cAMP is an important stimulator of PRL transcription in primary human T lymphocytes. Inhibition of both protein kinase A (PKA) and p38 MAPK partially abrogated cAMP-induced PRL expression. In addition, cAMP-induced phosphorylation of p38 was shown to occur independently of PKA and could be mimicked by a methylated cAMP analogue which specifically activates the recently discovered cAMP receptor EPAC (exchange protein directly activated by cAMP). Our findings suggest that cAMP induces PRL expression in T lymphocytes via cooperation of at least two different signaling pathways: a PKA-dependent pathway leading to the phosphorylation of cAMP response element-binding protein, and a PKA-independent pathway leading to p38 phosphorylation.

Transcriptional response to ionizing radiation in lymphocyte subsets.

Human lymphocyte subpopulations differ in their cellular responses to ionizing radiation. To shed light on the molecular basis of this effect, we characterized the transcriptional response to 1 Gy X-rays of CD4+ T lymphocytes. Of 18,433 genes tested, 102 were modulated more than 1.5-fold. The majority of the strongly activated genes were p53 targets involved in DNA repair and apoptosis. The expression of three of these genes was further tested by quantitative RT-PCR in lymphocyte subpopulations [CD4+ and CD8+ T, CD19+ B, CD56+ natural killer cells and peripheral blood lymphocytes (PBLs)] from ten adult donors. In contrast to DDB2, TNFRSF10B and BAX were differentially modulated among the subpopulations and the PBLs, being more activated in irradiated CD19+ B and CD8+ T lymphocytes. The level of BAX activation in the various subpopulations correlated with the sensitivity of the cells to radiation, suggesting its possible role in the differential radiosensitivity of hematopoietic cell subsets.

Effect of ionizing radiation on gene expression in CD4+ T lymphocytes and in Jurkat cells: unraveling novel pathways in radiation response.

To better understand at the molecular level the effect of ionizing radiation in leukocytes, the global transcriptional response to X-ray irradiation was studied in human CD4+ T lymphocytes and in Jurkat cells. Microarray analysis performed on freshly isolated human CD4+ lymphocytes 8 h after an LD50 irradiation dose of 1 Gy revealed that out of 13,825 genes, 1084 were modulated more than 1.5-fold. The most strongly up-regulated genes were predominantly p53 targets. In contrast, exposure of the CD4+ T lymphocyte-derived Jurkat leukemic cell line (with no functional p53 gene) to an equivalent LD50 dose (0.5 Gy) induced a partly different and more limited set of genes. Interestingly, this set of genes belonged to the Rho and cytokine signaling pathways regulated by low-dose ionizing radiation.

Gene expression induced by X-ray irradiation in human blood cell lines.

The response to X-ray irradiation of three different human hematopoietic cell lines originating from T (Jurkat), B (Raji), and promyelocytic (HL60) leukemia was analyzed. The survival after irradiation differed among the three cell lines, with Jurkat cells being the most vulnerable and HL60 being the least sensitive. The profile of gene expression was studied with the microarray technique in both Jurkat and HL60 cell lines. Out of the 13,800 different genes spotted on microarrays, very few genes (<0.5%) appeared to be induced more than 2-fold or repressed more than 2.5-fold in both cell lines.

Cytokine-like effects of prolactin in human mononuclear and polymorphonuclear leukocytes.

Some biochemical events following the binding of prolactin (PRL) to its receptor in normal human leukocytes were investigated. PRL enhanced JAK2 phosphorylation in peripheral blood mononuclear cells (PBMC) but not in granulocytes. PRL also induced phosphorylation of Stat-5 in PBMC and Stat-1 in granulocytes. Subsequent binding of Stat-5- and of Stat-1-like molecules to a GAS responsive element from the beta-casein promoter was detected by EMSA. p38 MAPK (but not p42/p44 MAPK) was activated by PRL in both leukocyte populations. PRL induced iNOS and CIS mRNA expression in granulocytes. Increased expression of IRF-1 and SOCS-2 was observed in granulocytes and of SOCS-3 and iNOS in PBMC. Similar effects were obtained with ovine and human PRL. Antiserum to PRL reduced iNOS and IRF-1 expression induced by PRL in granulocytes and reduced iNOS expression in PBMC. Also, pretreatment of granulocytes with a p38 MAPK inhibitor (SB 203580) prevented in part PRL-induced iNOS and IRF-1 expression. In PBMC, the p38 inhibitor decreased PRL-induced iNOS gene expression. These results indicate that PRL-induced gene regulation in leukocytes requires the activation of at least two different pathways: the Stat and the MAP kinase pathways. Moreover, although PRL activates Stat in both leukocyte types, signal transduction is different in granulocytes and in PBMC. Most importantly, PRL modulates the expression of genes crucial to leukocyte function. The present findings reinforce the concept that PRL has "cytokine-like" activity in human leukocytes.

Effects of prolactin on signal transduction and gene expression: possible relevance for systemic lupus erythematosus.

Receptors for prolactin (PRL-R) are expressed in normal leukocytes from rat and man. PRL signals through PRL-R associated Janus tyrosine kinase (Jak)-2 and signal transducers and activators of transcription (Stat). In addition, in human leukocytes PRL also activates the p38 MAP kinase pathway. PRL, at physiological concentrations, stimulates the expression of the interferon regulatory factor (IRF)-1 gene in rat spleen and bone marrow cells. In man, genes induced by PRL include several members of the 'suppressors of cytokine signaling' (SOCS) family and inducible nitric oxide synthase (iNOS; in mononuclear cells and in granulocytes) and IRF-1 (in granulocytes). Thus, in normal leukocytes, PRL induces the expression of several genes relevant to innate and acquired immune responses. Sex hormones, such as estrogen and PRL, have been implicated in the pathogenesis of murine and human SLE. Also defective signaling in leukocytes is a feature of the disease. What the origin is of aberrant signaling processes in SLE lymphocytes and how they relate to tolerance breakdown and immunopathology is still unknown. It is not unlikely that PRL is a player at some level. The exact contribution of PRL to immune responses in normal subjects and in SLE patients is not known. Further work should also indicate whether PRL might contribute to the onset or progression of the disease and assess the possible benefits of manipulating PRL concentrations in patients.

Tyrosine hydroxylase, but not dopamine beta-hydroxylase, is increased in rat frontal cortex after traumatic brain injury.

Chronic frontal lobe functional deficits after traumatic brain injury (TBI) may be associated with altered catecholamine systems in the frontal cortex. To test this, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) levels were examined by immunohistochemistry and Western blot at 1, 7, 14, and 28 days after TBI or sham surgery. No alterations in DBH levels were observed by Western blot at any time point examined, but there was a significant increase in TH expression 28 days after TBI (optical density 334 +/- 68% or 3.3-fold, ipsilateral and 218 +/- 39% or 2.2-fold, contralateral) relative to the sham controls. The increase in TH may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and increase the efficiency of dopamine neurotransmission chronically after TBI.

Prolactin-induced expression of cytokine-inducible SH2 signaling inhibitors in human hematopoietic progenitors.

OBJECTIVE: The prolactin (PRL) receptor (PRLR) utilizes the JAK2/STAT-5 pathway and induces expression of cytokine-inducible SH2 (CIS)/JAK2 binding (JAB) signaling inhibitors. We and others recently showed that CIS-3 and JAB abolish PRLR-mediated JAK2 activation and STAT-5 activity, whereas CIS-1, CIS-2, and CIS-4 had a negligible effect. Human CD34(+) hematopoietic progenitors express PRLRs and respond to PRL in vitro by enhanced cytokine-induced colony formation. To assess the signaling mechanism(s) involved in PRL-mediated enhancement of hematopoiesis and to identify further the CIS/JAB targets for PRL-mediated cellular responses, we assayed the effect of PRL, alone or in the presence of interleukin-3 (IL-3), on activation of STAT-5 and expression of CIS/JAB RNA in human cord blood (CB) CD34(+) cells. MATERIALS AND METHODS: Isolated CB CD34(+) cells were incubated in serum-free cultures in the absence or presence of recombinant human (rh)PRL, rhIL-3, or both. Cell lysates were subjected to Western blot analysis with anti-STAT-5 and anti-phospho-STAT-5 antibodies. Isolated RNA was subjected to semiquantitative reverse transcriptase polymerase chain reaction analysis of CIS/JAB expression. RESULTS: STAT-5 tyrosine phosphorylation was similarly induced by PRL and IL-3, with an additive effect detected in the presence of both stimuli. Both PRL and IL-3, alone or combined, failed to induce CIS-3 or JAB RNA expression in CD34(+) cells. Interferon-gamma had no effect on CIS-3/JAB induction in these cells. However, CIS-1 was induced by PRL < IL-3 < PRL+IL-3, whereas CIS-2 expression was induced by PRL = IL-3 < PRL+IL-3. CONCLUSIONS: Our findings show that PRL induces activation of STAT-5 and expression of similar CIS/JAB family members as IL-3 does in human CB CD34(+) cells. Because CIS-1 abolishes STAT-5 activation via the IL-3 but not the PRL receptor, the hematopoietic growth-promoting effects of PRL may involve its capacity to provide sustained STAT-5-mediated stimulatory signals to the cells.

Prolactin activates interferon regulatory factor-1 expression in normal lympho-hemopoietic cells.

It has been proposed that prolactin (PRL) is a lympho-hemopoietic growth and differentiation factor. We show here by Western blotting that PRL-receptors (PRL-R) are expressed in normal rat bone marrow and spleen cells. We also show that PRL stimulates the phosphorylation of the PRL-R-associated Janus tyrosine kinase (JAK)-2 in rat bone marrow and spleen cells. This leads to the activation and subsequent binding of signal transducer and activator of transcription (Stat) 5b to an interferon regulatory factor-1 (IRF-1) gamma activation sequence (GAS) as visualized by electromobility shift assay. As shown after reverse transcription of mRNA by polymerase chain reaction, PRL, at physiological concentrations (0.01 microg/ml), stimulates the expression of the IRF-1 gene in these normal cells. PRL could thus affect several aspects of the immune response.

Limited effects of placental and pituitary growth hormone on cytokine expression in vitro.

The hypothesis that growth hormone (GH) can affect immune responses in man has been evaluated by monitoring cytokine expression in cultures from peripheral blood mononuclear cells, by enzyme-linked immunosorbent assay (ELISA) and ribonuclease protection assay, and in tonsillar cells by ELISA. In addition to pituitary GH (GH-N), the placental form (GH-V), differing from pituitary GH by 13 amino acids has also been tested. Only few effects reached statistical significance and were in no case greater than 15%. Pituitary GH slightly reduced IL-5 production and stimulated IFN-gamma production. The latter effect was also observed with prolactin and could thus be induced through the prolactin receptor. It is proposed that GH has no strong effects on the parameters investigated, possibly as a result of redundancy in the cytokine network. Alternatively, effects on leukocytes are mediated by other tissues such as the liver or are clear only in response to stronger challenges.

Myeloid leukemic cells express and secrete bioactive pituitary-sized 23 kDa prolactin.

Prolactin (PRL) is a 23 kDa polypeptide hormone of pituitary origin which is of major importance for reproduction. In addition, PRL has immunomodulatory effects and can be produced in small quantities in nonpituitary tissues. To address possible autocrine or paracrine functions of PRL in leukemia, we characterized immunoreactive PRL from the culture medium of leukemic cells. The myeloid cell line Eol-1 expresses the long extrapituitary type mRNA for PRL and synthesizes immunoreactive PRL with a molecular weight of 23 kDa. The biological activity in Eol-1 culture medium was determined using the Nb2 bioassay. This activity co-eluted with recombinant human (rh) PRL on an S-200 Sephacryl gel filtration column and could be blocked by anti-PRL antiserum. Western blot analysis and Nb2 bioassays also suggest that acute myelogenous leukemic blasts secrete bioactive 23 kDa PRL in one out of three tested patients.

Expression of SOCS genes in normal and leukemic human leukocytes stimulated by prolactin, growth hormone and cytokines.

To evaluate the possible role of the recently described family of suppressors of cytokine signaling (SOCS) factors in the human lympho-hemopoietic system, we have monitored SOCS factor expression, both constitutive and induced by either cytokines, prolactin (PRL) or growth hormone (GH), using polymerase chain reaction in normal and leukemic cells. CIS (cytokine-inducible SH2-containing protein), SOCS-2 and SOCS-3 were constitutively expressed in peripheral blood mononuclear leukocytes. SOCS-3 expression was enhanced by PRL or by IFN-gamma. In bone marrow cells and granulocytes, CIS expression was induced and SOCS-2 enhanced by IFN-gamma and by PRL. In tonsillar cells, CIS expression was increased and SOCS-2 was induced by IL-1beta, IL-6, PRL and GH. SOCS-3 expression was enhanced by IL-1beta. The expression of SOCS-7 was increased by IL-6, PRL and GH. In Raji B-lymphoma cells, the expression of SOCS-2 and SOCS-7 was enhanced by IL-1beta. In THP-1 myeloid leukemia cells pretreated with TPA (to induce receptors for IFN-gamma), IFN-gamma induced SOCS-2. Jurkat cells expressed more SOCS-2 when exposed to PRL. Original observations in this work include the first report on SOCS-7 induction by cytokines. Also our data shed new light on the possible involvement of PRL and GH in the cytokine network. These hormones could modulate the transduction of signals originating from receptors for various cytokines.

Effects of selected herbicides on cytokine production in vitro.

To evaluate possible deleterious effects of commonly used herbicides on leukocytes, cytokine production was selected as a sensitive indicator. After in vitro exposure of human peripheral blood mononuclear cells from normal donors, the production of all 3 cytokines tested--interferon-gamma (a type 1 cytokine), interleukin-5 (a type 2 cytokine) and tumor necrosis factor-alpha (an inflammatory cytokine)--was impaired by up to 70, 50 and 70% respectively in a concentration-dependent manner in cultures exposed to atrazine (0.03-3 microM in 1% dimethylsulfoxide, DMSO). The effect paralleled that seen with dexamethasone, a known immunosuppressive agent. Other pesticides also dissolved in DMSO--mecoprop, simazine or MCPA (each up to 1 microM)--or dissolved in phosphate-buffered saline--diuron (up to 1 microM), isoproturon (up to 3 microM), metoxuron (up to 8 microM) or metamitron (up to 80 microM)--showed no concentration-related effects on cytokine production. There was, however, an inhibition of IFN-gamma and TNF-alpha production by simazine, metoxuron and mecoprop and of all three cytokines tested by diuron. MCPA (0.01 and 0.1 microM) stimulated the production of TNF-alpha. Thus, exposure to herbicides leading to plasma levels in the micromolar range induces imbalance in cytokine production.

Growth hormone and prolactin expression in the immune system.

Prolactin (PRL) and growth hormone (GH) are pituitary hormones that play pivotal roles in lactation and body growth, respectively. In addition, both hormones have been implicated as modulators of immune responses. Since the expression of GH and PRL by leukocytes points to autocrine or paracrine roles during immune responses, our study is aimed at PRL- and GH-production in leukocytes. We show that human peripheral blood granulocytes, which express GH and PRL mRNA, contain high molecular-weight immunoreactive variants of GH and PRL (37 and 43 kDa, respectively), but not the pituitary-sized hormones. Secretion of these variants, or biologically active material as assessed by the Nb2 bioassay, was not detected. On the other hand, certain leukemic myeloid cells secrete 23-kDa, pituitary-sized, PRL, which is biologically active.

Differential actions of the dopamine agonist bromocriptine on growth of SMtTW tumors exhibiting a prolactin and/or a somatotroph cell phenotype: relation to dopamine D2 receptor expression.

Dopamine (Da) and Da agonists are known to inhibit secretion and proliferation of normal and tumoral PRL cells, through receptors of D2 subtype. Because of the lack of an experimental model, the relationship between bromocriptine (BR) sensitivity and D2 receptor expression is poorly documented. Such a relationship was analyzed using five lineages of spontaneous transplantable rat pituitary tumors (SMtTW) exhibiting different PRL/GH phenotypes. From plasma PRL and GH concentrations of rats bearing the tumors and tumor messenger RNA contents, tumors were classified as PRL (SMtTW2), somatotroph (SMtTW10), or somatomammotroph (SMtTW5) tumors. Two lineages (SMtTW3 and SMtTW4) represented variants producing PRL and GH but with a high predominance of PRL. With the exception of SMtTW4 tumors, which were malignant, all the tumors were benign and differed in their growth rate. Hormone production and growth of tumors with a PRL or a somatomammotroph phenotype were reduced by about 90% under BR treatment, whereas somatotroph tumors and the PRL malignant tumors were totally insensitive to BR. D2 receptor messenger RNA was present in all BR-sensitive tumors and was not detected in BR-resistant tumors. In conclusion, using five lineages of SMtTW tumors that are representative of the most frequent tumors encountered in human pituitary pathology, we found a full concordance between tumor responses to BR and the expression of D2 receptor by the tumors. The identification of a tumor lineage with a malignant phenotype, secreting high amounts of PRL and presenting a resistance to BR, supports the idea that Da-resistant prolactinomas are aggressive tumors.

Prolactin, growth hormone and the immune system in humans.

Prolactin (PRL) and growth hormone (GH) qualify as lymphoid growth and differentiation factors. Yet, long-standing hyper- or hyposecretion of PRL or GH does not induce any significant alteration of the immune system. Subclinical changes in laboratory parameters (such as chemotaxis or phagocytosis by granulocytes or macrophages or natural killer cell activity) have been reported in some of these conditions. The GH-insulin-like growth factor (IGF)-I axis is dysregulated in ageing, in catabolic states and in critical illness. Immune parameters, such as infection rate, are being monitored during clinical trials with GH or IGF-I. Hyperprolactinaemia may play an aggravating role in systemic lupus erythematosus, in autoimmune thyroiditis and in other autoimmune diseases. The patient may benefit from increased alertness about interactions between the endocrine and immune systems.

Pituitary growth hormone release and gene expression in cafeteria-diet-induced obese rats.

In human obesity as well as in rat obesity models a decrease in spontaneous and stimulated GH secretion has been a constant finding. The presence of a decreased pituitary GH synthesis in diet-induced obese male rats was investigated and its possible relationship with obesity-related changes in peripheral hormones was analyzed. Cafeteria-diet-overfed obese male Wistar rats with body fat percentage above 30% had a significantly decreased pituitary GH mRNA transcript level assessed by both Northern blot and in situ hybridization, and a lower pituitary GH protein level as demonstrated by immunocytochemistry. The GH transcript level correlated negatively with the serum leptin and positively with the IGF-I concentration. No differences in circulating tri-iodothyronine, non-fasting insulin and corticosterone levels were found between overfed and control rats. GH release by cultured pituitary cells from overfed rats was comparable to that by cells prepared from control rats. In contrast, incubation of normal pituitary cells with serum from overfed rats for 3 days gave a significantly lower GH release than after incubation with serum from non-obese rats. In conclusion, cafeteria-diet-induced obese male Wistar rats have a decreased pituitary GH gene expression and a modifiable GH release in in vitro experiments. A possible role for peripheral circulating factors, like leptin and IGF-I, in decreasing the pituitary GH synthesis and release in obese rats is discussed.

Nuclear factor 1 regulates the distal silencer of the human PIT1/GHF1 gene.

Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2. 3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region.