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C Clapp, FJ Lopez-Gomez, G Nava, A Corbacho, L Torner, Y Macotela, Z Duenas, A Ochoa, G Noris, E Acosta, E Garay and G Martinez de la Escalera

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.

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L Torner, G Nava, Z Duenas, A Corbacho, S Mejia, F Lopez, M Cajero, G Martinez de la Escalera and C Clapp

Estrogens are recognized regulators of the expression of neurohypophyseal hormones and of anterior pituitary prolactin (PRL). Here we have investigated whether the levels of PRL mRNA and of 23 and 14 kDa PRL variants present in the hypothalamo-neurohypophyseal system change during the estrous cycle or in response to estrogen treatment. The reverse transcription polymerase chain reaction (RT-PCR) was performed to examine PRL mRNA expression in isolated paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei. In both nuclei PRL mRNA levels appeared higher in cycling females than in male rats, with the highest level occurring at estrus. This increase may involve estrogen action, since estrogen administration to ovariectomized rats was associated with apparently higher PRL mRNA levels in both the PVN and SON. Expression of the PRL gene at these sites may occur via both transcriptional factor Pit-1-dependent and -independent mechanisms. RT-PCR detected the mRNA for Pit-1 in the PVN but only at estrus. The concentration of the 23 kDa immunoreactive PRL determined in the neurohypophysis was significantly higher during estrus and after estrogen treatment. However, no difference was detected in the levels of the neurohypophyseal 14 kDa PRL-like fragment along the estrous cycle nor after estrogen administration. This lack of parallelism between neurohypophyseal PRLs could relate to an estrogen-induced inhibition of the proteolysis of 23 kDa PRL at this site, since estrogen treatment reduced the activity of neurohypophyseal proteolytic enzymes able to cleave PRL. Altogether our results are consistent with estrogens having a stimulatory effect on PRL gene expression in the hypothalamo-neurohypophyseal system and a concomitant inhibitory action on PRL proteolysis at this site.

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AM Corbacho, Y Macotela, G Nava, L Torner, Z Duenas, G Noris, MA Morales, G Martinez De La Escalera and C Clapp

Members of the prolactin (PRL) hormonal family have direct effects on endothelial cell proliferation, migration and tube formation. Moreover, isoforms of PRL may function as autocrine regulators of endothelial cells. Bovine brain capillary endothelial cells (BBCEC) express the PRL gene, while anti-PRL antibodies inhibit BBCEC proliferation. Here, we show the expression of the PRL gene into various PRL isoforms in endothelial cells from the human umbilical vein. Reverse transcription-polymerase chain reaction of total RNA from human umbilical vein endothelial cells (HUVEC) detected the full-length PRL mRNA as well as a 100 bp smaller PRL transcript similar to the one previously reported in BBCEC. HUVEC were positive to PRL immunocytochemistry. In addition, various PRL immunoreactive proteins were detected in HUVEC extracts and HUVEC conditioned media by metabolic labelling immunoprecipitation analysis. These PRL immunorelated proteins had apparent molecular masses of 60, 23, 21, 16 and 14 kDa. In contrast to previous findings in BBCEC, HUVEC conditioned media contained very little PRL bioactivity as determined by the selective bioassay of Nb2 cell proliferation. Moreover, some polyclonal or monoclonal antibodies directed against PRL stimulated HUVEC proliferation, in contrast to the inhibitory effect seen in BBCEC. The present findings extend the previous observations about the expression of PRL gene in endothelial cells from bovine brain capillaries to human cells of the umbilical vein, implicating that endothelium from different types of vessels and species share the expression of PRL gene but may differ in the putative autocrine role of the PRL isoforms expressed.