The 23-kDa form of prolactin (PRL) has been proposed to function as both a mature hormone and a prohormone precursor for different uniquely bioactive forms of the molecule. We have shown that the 16-kDa N-terminal fragent of PRL (16K PRL) inhibits angiogenesis via a specific receptor. In addition, 16K PRL stimulates natriuresis and diuresis In the rat, and kidney membranes contain high-affinity specific binding sites for this PRL fragent. 16K PRL can be derived from an enzymatically cleaved form of PRL (cleaved PRL). With the use of a specific 16K PRL antiserum, we have loalid a 14-kDa immunoreactive protein in the paraventricular and supraoptic nuclei of the hypothalamus and in the neurohypophysis. Reverse transcription-polymerase chain reaction of RNA from isolated paraventricular nuclei showed the expression of the full-length PRL mRNA. The neurohypophysis was found to contain the enzymes that produce cleaved PRL, small amounts of PRL, and cleaved PRL. Medium conditioned by neurohypophyseal cultures, enriched with the 14-kDa immunoreactive protein, has antiaogenic effects that are blocked by the 16K PRL antiserum. These results are consistent with the expression of PRL in the hypothamicneurohypophyseal system, and the preferential processing of the protein into a 14-kDa gent with biological and immunological properties of 16K PRL.
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 is 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 basic fibroblast growth-factorstimulated 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.
We have described the expression of the prolactin (PRL) gene and the occurrence of PRL-like immunoreactive proteins in the hypothalamic-neurophypophyseal system of the rat. Here, we investigated the nature of neurohypophyseal PRL-like antigens, by studying the biological activity of medium conditioned by incubated neurohypophyses in specific bioassays for PRL and for the 16 kDa N-terminal fragment of PRL (16K PRL). Neurohypophyseal conditioned medium (NHCM) obtained after incubating neurohyphyseal lobes (1 h at 37°C) was enriched with proteins of 14 kDa and 23 kDa, that crossreacted with PRL-and 16K PRL-directed antisera. The NHCM stimulated in a dose-dependent fashion the proliferation of Nb2-lymphoma PRL-dependent cells. This effect paralleled that of PRL and 16K PRL standards and was neutralized by different dilutions of both PRL-and 16K PRL-antisera. Also, the NHCM inhibited the proliferation of endothelial cells in culture, an antiangiogenic-effect exerted by 16K PRL. The antiangiogenic effect of the NHCM was parallel to that of 16K PRL standard and neutralized by 16K PRL antiserum in a dose-dependent fashion. These results indicate that NHCM contains proteins that share receptor activation properties as well as antigenic determinants with both PRL and 16K PRL.
The recently described expression of the PRL gene, and the occurrence of a 14-kilodalton (kDa)PRL-like immunoreactive protein in the hypothalamo-neurohypophyseal system of the rat have raised the possibility that PRL variants are released from neurohypophyseal terminals into the blood. In this study, we investigated the local production of a hypothalamo-neurohypophyseal 14-kDa PRL-like protein by showing an independent origin from adenohypophyseal PRL. No 14-kDa PRL-like protein was detected in adenohypophyseal extracts by Western blots, whereas chronic hypophysectomy produced no change in the immunocytochemical detection of PRLs in supraoptic and paraventricular magnocellular neurons. In addition, a 14-kDa immunoreactive PRL-like protein was released into the medium by incubated neurohypophyseal lobes. Western blot analysis showed that significantly more of this 14-kDa protein was released into calcium-containing medium (1.8 mM) than into calcium-free medium. Furthermore, depolarizing concentrations of potassium (56 mM) increased by 3-fold the release of immunoreactive PRL by incubated hypothalamo-neurohypophyseal explants. In addition, a 14-kDa PRL-like antigen was detected in the circulation of the rat by Western blot analysis. These results are consistent with the local synthesis and calcium-dependent release of neurohypophyseal PRL-like proteins that include a predominant 14-kDa form.
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