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AM Corbacho, G Martinez De La Escalera, and C Clapp

Prolactin, growth hormone and placental lactogen are members of a family of polypeptide hormones which share structural similarities and biological activities. Numerous functions have been attributed to these hormones, among which stand out their recently discovered effects on angiogenesis, the process by which new blood vessels are formed from the pre-existing microvasculature. Prolactin, growth hormone and placental lactogen, along with two non-classical members of the family, proliferin and proliferin-related protein, can act both as circulating hormones and as paracrine/autocrine factors to either stimulate or inhibit various stages of the formation and remodeling of new blood vessels, including endothelial cell proliferation, migration, protease production and apoptosis. Such opposing actions can reside in similar but independent molecules, as is the case of proliferin and proliferin-related protein, which stimulate and inhibit angiogenesis respectively. The potential to exert opposing effects on angiogenesis can also reside within the same molecule as the parent protein can promote angiogenesis (i.e. prolactin, growth hormone and placental lactogen), but after proteolytic processing the resulting peptide fragment acquires anti-angiogenic properties (i.e. 16 kDa prolactin, 16 kDa growth hormone and 16 kDa placental lactogen). The unique properties of the peptide fragments versus the full-length molecules, the regulation of the protease responsible for specific protein cleavage, the selective expression of specific receptors and their associated signal transduction pathways are issues that are being investigated to further establish the precise contribution of these hormones to angiogenesis under both physiological and pathological situations. In this review article, we summarize the known and speculative issues underlying the effects of the prolactin, growth hormone and placental lactogen family of proteins on angiogenesis, and address important remaining enigmas in this field of research.

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C Zermeño, J Guzmán-Morales, Y Macotela, G Nava, F López-Barrera, J B Kouri, C Lavalle, G Martínez de la Escalera, and C Clapp

The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

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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.

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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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Y Macotela, C Mendoza, AM Corbacho, G Cosio, JP Eiserich, A Zentella, G Martinez de la Escalera, and C Clapp

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-kappaB), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-kappaB in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-kappaB. Finally, treatment with 16K PRL induced the degradation of the NF-kappaB inhibitor kappaB-beta (IkappaB-beta), and such degradation was prevented by blocking IkappaB-beta phosphorylation. Altogether, these results show that 16K PRL activates NF-kappaB nuclear translocation via the phosphorylation and degradation of IkappaB-beta. These findings are consistent with NF-kappaB being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

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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.