Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers.

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.

The prolactin (PRL) gene is expressed in the hypothalamo-neurohypophyseal system as revealed by the detection of the PRL mRNA and of PRL-like immunoreactive and biologically active proteins in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and in the neurohypophysis. We have investigated the distribution of cells containing PRL-like molecules in the PVN and SON by immunocytochemistry with a specific antiserum directed against the 16-kD N-terminal fragment of PRL. PRL-positive cells were found to be concentrated throughout the ventral SON and in the lateroposterior region of the PVN. The cellular distribution of PRL-immunoreactive cells resembled more closely that of vasopressin (VP) than that of oxytocin magnocellular neurons. Moreover, double immunofluorescence labelling, followed by confocal microscopy, indicated the coexistence of PRL- and VP-related antigens within the same neurons of the PVN and SON. Pre-embedding immunoperoxidase on the ultrastructural level showed a PRL-like product in granular-type particles within the neural soma and projections in the SON and PVN. These findings are consistent with the expression and secretion of PRL-like molecules by vasopressinergic neurons of the hypothalamo-neurohypophyseal system.

Activity of the magnocellular neurons that synthesize vasopressin and oxytocin in the paraventricular and supraoptic nuclei of the hypothalamus can be modulated by local release of neuromediators within the nuclei. Among the bioactive peptides that may play autocrine or paracrine roles in this system is prolactin (PRL). Paraventricular and supraoptic neurons express PRL mRNA and contain and secrete PRL-like proteins of 23 and 14 kDa. We investigated the localization of PRL receptors in vasopressinergic and oxytocinergic magnocellular neurons using dual-label immunofluorescence. The results demonstrate that both vasopressin- and oxytocin-immunoreactive cells of the paraventricular and supraoptic nuclei contain the PRL receptor. In addition, we investigated the possible regulation of vasopressin secretion by PRL using hypothalamo-neurohypophyseal explants in culture. The results show that PRL and a 16 kDa N-terminal fragment of the hormone that is analogous to the neurohypophyseal 14-kDa PRL fragment stimulate the release of vasopressin. Together, these findings support the hypothesis that vasopressinergic and oxytocinergic neurons of the magnocellular secretory system are regulated directly by various isoforms of PRL via autocrine/paracrine mechanisms.

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.

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.

Insulin resistance in the vasculature is a characteristic feature of obesity and contributes to the pathogenesis of vascular dysfunction and disease. However, the molecular mechanisms underlying obesity-associated vascular insulin resistance and dysfunction remain poorly understood. We hypothesized that TRAF3IP2 (TRAF3 interacting protein 2), a proinflammatory adaptor molecule known to activate pathological stress pathways and implicated in cardiovascular diseases, plays a causal role in obesity-associated vascular insulin resistance and dysfunction. We tested this hypothesis by employing genetic-manipulation in endothelial cells in vitro, in isolated arteries ex vivo, and diet-induced obesity in a mouse model of TRAF3IP2 ablation in vivo. We show that ectopic expression of TRAF3IP2 blunts insulin signaling in endothelial cells and diminishes endothelium-dependent vasorelaxation in isolated aortic rings. Further, 16 weeks of high fat/high sucrose feeding impaired glucose tolerance, aortic insulin-induced vasorelaxation, and hindlimb postocclusive reactive hyperemia, while increasing blood pressure and arterial stiffness in wild-type male mice. Notably, TRAF3IP2 ablation protected mice from such high fat/high sucrose feeding-induced metabolic and vascular defects. Interestingly, wild-type female mice expressed markedly reduced levels of TRAF3IP2 mRNA independent of diet and were protected against high fat/high sucrose diet-induced vascular dysfunction. These data indicate that TRAF3IP2 plays a causal role in vascular insulin resistance and dysfunction. Specifically, the present findings highlight a sexual dimorphic role of TRAF3IP2 in vascular control and identify it as a promising therapeutic target in vasculometabolic derangements associated with obesity, particularly in males.