Post-stroke treatment with 17ß-estradiol exerts neuroprotective effects in both normotensive and hypertensive rats.

Although ischemic stroke is a major cause of death worldwide and the predominant cause of acquired disability, the only effective drug therapy that has been developed thus far is reperfusion by tissue plasminogen activator. Since most patients do not qualify for this treatment, new methods have to be developed. It is well known that estradiol (E2) exerts neuroprotective effects in different models of cerebral ischemia, but post-stroke treatment after an acute stroke has hardly been investigated. As many patients with an acute ischemic stroke have arterial hypertension, it is also of interest to evaluate the influence of this co-morbidity on the treatment efficacy of E2. The effects of E2 administered 30min after a transient middle cerebral artery occlusion (tMCAO) induced by an intracerebral injection of endothelin-1 were assessed in male normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Treatment with E2 reduced infarct size in both WKY and SHRs and decreased the number of degenerating neurons, indicating that acute treatment with E2 is indeed neuroprotective. To address the role of glia in neuroprotection, the effects of E2 on the activation of microglia and astrocytes was determined. It appeared that E2 had no effect on microglial activation, but reduced the activation of astrocytes in SHRs but not in the normotensive controls. We conclude that post-stroke E2 treatment in both normotensive and hypertensive rats is neuroprotective. Although the presence of hypertension changed the astrocytic response to E2, it did not affect treatment efficacy.

Attenuation of IGF-I receptor signaling inhibits serum-induced proliferation of prostate cancer cells.

OBJECTIVE: Several studies showed that high serum levels of insulin-like growth factor-I (IGF-I) correlate with an increased risk for prostate cancer, although the causal role of IGF-I remains to be established. In this study, we addressed the role of IGF-I as a serum factor on the growth of two androgen-independent cell lines (Du145 and PC3) and one androgen-dependent cell line (LNCaP). DESIGN: We investigated the effects of a blocking antibody against the IGF-I receptor (αIR3) on DNA synthesis in prostate cancer cells cultured in the presence of recombinant human IGF-I or normal human serum (NHS). RESULTS: We show that in all three prostate cancer cell lines, NHS exerts a markedly stronger stimulating effect on DNA synthesis than IGF-I, and that the effect of NHS can be completely abrogated by an antibody against the IGF-I receptor (αIR3). Using pharmacological inhibitors of the two canonical IGF-I receptor signaling pathways, we show that the phosphatidylinositol-3'-kinase (PI3K) and the Mek-Erk pathways are not required for the stimulating effect of NHS. CONCLUSION: Our observations indicate that the stimulating effect of NHS is completely dependent on IGF-I receptor signaling transduction and that IGF-I stimulates DNA synthesis in prostate cancer cells in strong synergy with other serum factors. We speculate that the role of other serum factors could explain the discrepancy between the results observed in different animal models to study the function of IGF-I in prostate cancer.

Modulation of cytokine production by cyclic adenosine monophosphate analogs in human leukocytes.

Cyclic adenosine monophosphate (cAMP) is a well-known second messenger that operates through different signaling molecules, including protein kinase A (PKA) and guanine exchange proteins directly activated by cAMP (EPAC). Cell-permeable cAMP analogs such as 8-(4-chloro-phenyl-thio)-cAMP (8-pCPT-cAMP) modulate cytokine secretion by different leukocyte subsets, including T cells and monocytes. Since cAMP-modulating drugs such as phosphodiesterase inhibitors are being tested in inflammatory disorders such as asthma and chronic obstructive lung disease, it is important to obtain more insight into the regulation of cytokine production by cAMP. To address the signaling molecules involved in cAMP-mediated modulation of cytokine production, we used cAMP derivatives such as N(6)-benzoyladenosine-cAMP (6-Bnz-cAMP) and 8-pCPT-2-O-methyl cAMP (8-pCPT-2'-O-Me-cAMP), which selectively activate either PKA or EPAC, respectively. We show that in T cells, 6-Bnz-cAMP exerts similar globally inhibiting effects on cytokine secretion as 8-pCPT-cAMP, indicating that these effects are mediated by PKA. On the contrary, 8-pCPT-2'-O-Me-cAMP specifically inhibits the production of interleukin-10 (IL-10) in lipopolysaccharide-activated T-cell-depleted peripheral blood mononuclear cells, whereas the production of IL-1ß, tumor necrosis factor α, and IL-12 is not or hardly affected. Inhibition by 8-pCPT-2'-O-Me-cAMP of IL-10 production was confirmed using purified monocytes. Further, in B cells 6-Bnz-cAMP, but not 8-pCPT-2'-O-Me-cAMP, stimulated IL-10 production. In conclusion, cAMP stimulates IL-10 production via PKA in activated B cells, but inhibits IL-10 production in activated monocytes through EPAC. We speculate that selective effects of PKA and EPAC on cytokine production in leukocyte subsets open up therapeutic possibilities using selective activators or inhibitors of EPAC or PKA.

Tumor necrosis factor-alpha activates the extrapituitary PRL promoter in myeloid leukemic cells.

The pituitary hormone prolactin (PRL) is also produced extrapituitarily by cells of the immune system. In leukocytes PRL expression is directed by an alternative promoter, located 5800 bp upstream of the pituitary promoter. We have shown here that this alternative promoter is activated in myeloid leukemic cells by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. The effect of TNF-alpha on promoter activation was blocked by the protein kinase C (PKC)-inhibitor GFX109203. In addition, we have shown that the TNF-alpha-responsive region is located between -1842 and -1662 of the extrapituitary PRL promoter. Our findings might have clinical relevance in view of recent data indicating leukocyte-derived PRL is involved in auto-immune and hematological disorders.

Modulation of prolactin expression in human T lymphocytes by cytokines.

Besides its pivotal role in reproduction, the polypeptide hormone prolactin (PRL) has immunomodulatory properties. Whereas the bulk of circulating PRL is produced by the pituitary, PRL is also produced by the decidua, the myometrium, the mammary gland and leukocytes. Extrapituitary PRL expression is regulated differently from that in the pituitary, due to the use of an alternative promoter. Here we show for the first time that in T lymphocytes PRL expression is subject to regulation by cytokines. We established that both IL-2 and IL-4 reduced PRL mRNA levels in T lymphocytes to 25 and 28% of control values, respectively. PRL mRNA expression was inhibited to a lesser extent by IL-1beta, which decreased PRL mRNA levels to 58% of control values.

Multiple, PKA-dependent and PKA-independent, signals are involved in cAMP-induced PRL expression in the eosinophilic cell line Eol-1.

Besides its pivotal role in reproduction, the polypeptide hormone prolactin (PRL) has been attributed an immunomodulatory function. Extrapituitary PRL expression is regulated differently from that in the pituitary, due to the use of an alternative promoter. In leukocytes, cAMP is an important regulator of PRL expression. We report that in the human eosinophilic cell line Eol-1, cAMP-induced PRL expression is partially abrogated by two protein kinase A (PKA) inhibitors (H89, PKI) and by the p38 inhibitor SB203580. Phosphorylation of p38 was PKA-independent and could be stimulated by a methylated cAMP analogue, which specifically activates the exchange factor directly activated by cAMP (EPAC). Furthermore, cAMP induced a PKA-dependent phosphorylation of cAMP-responsive element binding protein (CREB). We postulate that cAMP induces PRL expression via two different signalling pathways: a PKA-dependent pathway leading to the phosphorylation of CREB, and a PKA-independent pathway leading to the phosphorylation of p38.

Mechanism of prostaglandin (PG)E2-induced prolactin expression in human T cells: cooperation of two PGE2 receptor subtypes, E-prostanoid (EP) 3 and EP4, via calcium- and cyclic adenosine 5'-monophosphate-mediated signaling pathways.

We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE(2) and that cAMP acts synergistically with Ca(2+) or protein kinase C on the activation of the upstream prolactin promoter. Using the transcription inhibitor actinomycin D, we now show that PGE(2)-induced prolactin expression requires de novo prolactin mRNA synthesis and that PGE(2) does not influence prolactin mRNA stability. Furthermore, PGE(2)-induced prolactin expression was inhibited by protein kinase inhibitor fragment 14-22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca(2+)-mediated signaling cascades. Using specific PGE(2) receptor agonists and antagonists, we show that PGE(2) induces prolactin expression through engagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE(2) induces an increase in intracellular cAMP concentration as well as intracellular calcium concentration via EP4 and EP3 receptors, respectively. In transient transfections, 3000 bp flanking the leukocyte prolactin promoter conferred a weak induction of the luciferase reporter gene by PGE(2) and cAMP, whereas cAMP in synergy with ionomycin strongly activated the promoter. Mutation of a C/EBP responsive element at -214 partially abolished the response of the leukocyte prolactin promoter to PGE(2), cAMP, and ionomycin plus cAMP.

Clinical and biological characterization of macroprolactinemia with and without prolactin-IgG complexes.

OBJECTIVE: Macroprolactinemia, which can be detected by a polyethylene glycol (PEG) precipitation test, is a clinically and biologically heterogeneous condition. In this study, we analyzed whether the clinical presentation, the hormonal findings and the in vitro lactogenic activity differed between macroprolactinemic patients with and without circulating prolactin (PRL)-IgG complexes. DESIGN: Clinical data were reviewed and additional hormonal studies were performed in 50 hyperprolactinemic patients with macroprolactinemia. METHODS: Macroprolactinemia was identified by a PRL recovery after PEG precipitation of <50%, as measured by an automated commercial immunoassay system and circulating PRL-IgG complexes by an abnormal PRL binding to anti-IgG agarose. RESULTS: PRL-IgG complexes were found in 46 patients. The origin of hyperprolactinemia in these 46 patients was idiopathic in 33 patients, while a pituitary lesion or stalk magnetic resonance imaging or computed tomography scan was detected in 13 patients found compression. Galactorrhea was found in 11 of these 46 patients, while this condition was present in three of the four patients without circulating PRL-IgG complexes. The median free PRL concentration was significantly lower in patients with PRL-IgG complexes than in the group without complexes (243 vs 969 mIU/l; P<0.005), whereas median total PRL immunoreactivity and median PRL bioactivity in the Nb2 assay were not significantly different. In patients with circulating PRL-IgG complexes, Nb2 bioassay results correlated significantly with total PRL immunoreactivity (r=0.64; P<0.0001), but not with free PRL results (r=0.24; P<0.17). CONCLUSIONS: These results indicate that PRL-IgG complexes (i) account for most cases of macroprolactinemia--as identified by PEG precipitation--in hyperprolactinemic patients presenting with a variety of diagnoses, (ii) are not associated with a specific clinical presentation, (iii) can be found in patients with diverse pituitary pathologies, and (iv) possess an in vitro lactogenic activity in the Nb2 bioassay in relation to their immunoreactivity.

Regulation of prolactin expression in leukemic cell lines and peripheral blood mononuclear cells.

To address the role of different intracellular signals in prolactin (PRL) expression in leukocytes, we have investigated the effects of chlorophenylthio-cAMP (cptcAMP), phorbol myristate acetate (PMA) and ionomycin on the activation of the upstream PRL promoter in several leukemic cell lines. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. In freshly isolated peripheral blood mononuclear cells (PBMC), PRL expression could only be stimulated by cptcAMP. The physiological importance of cAMP in the regulation of PRL expression in leukocytes is suggested by the finding that in PBMC, PRL expression is enhanced by prostaglandin-E(2) and the beta(2)-adrenergic agonist terbutaline, which both signal through cAMP.