Multiomics approach to identify novel biomarkers for dilated cardiomyopathy: Proteome and transcriptome analyses of 4C30 dilated cardiomyopathy mouse model.

Dilated cardiomyopathy (DCM) is an intractable disease, without any radical treatment option other than cardiac transplantation. Additionally, biomarkers to determine progressive staging are not yet available. Irrespective of the diversity of causative gene mutations, the phenotype of DCM is rather common. Therefore, it is plausible to determine DCM staging in terms of variations in protein and mRNA levels. In this study, we performed proteome and transcriptome analysis of the left ventricle of 4C30 DCM model mice showing mild and severe phenotypes at 12 and 24 weeks (wk) after birth, respectively. Proteomic analyses results showed 109 proteins that increased and 133 others that decreased among 1874 detected proteins. We selected biomarker candidates by confirming consistent alterations in protein levels at 12 and 24 wk, and mRNA levels at 12 wk, and narrowed these down based on the requirement that they should be detectable in blood. Finally, we selected six biomarker candidates based on sustained or augmented alteration at 24 wk and confirmed their definite alterations in the left ventricle by quantitative polymerase chain reaction, western blot analysis, and multiple reaction monitoring (MRM). To assess the validity of this strategy, we measured plasma concentrations of the six candidates by MRM method and identified two proteins (FTL1 and GRP78) that demonstrated significant elevation in the 4C30 mice plasma. Taken together, a multiomics strategy comparing tissue expression levels of proteins and mRNAs between diseased and control groups, with appropriate confirmation, is a promising approach for the discovery of new biomarkers. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 491-502, 2016.

Immunocytochemical localization of kisspeptin neurons in the rat forebrain with special reference to sexual dimorphism and interaction with GnRH neurons.

Kisspeptin/metastin has been implicated as a critical regulator in luteinizing hormone (LH) secretion and the reproductive system mediating the effect of estrogen on GnRH neurons. In the present study we examined the sex differences in the effects of estrogen on Kiss1/kisspeptin expression in the forebrain by using gonadectomized rats to assess the interaction of kisspeptin and GnRH neurons. Kiss1/kisspeptin cell bodies were abundant in the rostral periventricular area of the third ventricle (RV3P) and the arcuate nucleus (ARC). A few cell bodies were also observed in other portions of the forebrain, i.e. the bed nucleus of the stria terminalis (BST), the paraventricular hypothalamic nucleus (PaAP), the ventromedial hypothalamic nucleus (VMH), and the medial amygdaloid nucleus (MeA). Kisspeptin-immunoreactive fibers were found mainly in the median eminence (ME), the ARC, and the RV3P, but were scarce in the preoptic area (POA), where GnRH neurons are localized. We also found that estrogen triggers expression of the Kiss1 gene and peptide within all the regions except the ARC, and that the effects in the RV3P, BST, PaAP, and VMH are greater in estrogen treated ovariectomized female rat. It is noteworthy that kisspeptin and GnRH neurons were densely associated in the ME but were rarely in contact in the POA. Thus, our results suggest that kisspeptin-positive neurons, except for the ones in the ARC, are related not only to estrogen-positive feedback, but also sex dimorphism, and that kisspeptin regulates GnRH release in the ME rather than the POA.

Circadian transcriptional factor DBP regulates expression of Kiss1 in the anteroventral periventricular nucleus.

The expression of Kiss1 in the anteroventral periventricular nucleus (AVPV) and its product, metastin/kisspeptin, show a circadian pattern with a peak in the evening, which shows a strong phase relationship with the time of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge in rodents. Here we report that a circadian transcriptional factor, albumin D-site binding protein (Dbp), was able to trigger mKiss1 transcription via the D-box, and this effect was combined with those of estrogen receptor α (ERα) and its ligand, estrogen. A histological study demonstrated that some cells in the AVPV co-expressed Dbp with ERα in adult female rats. Expression of ERα was not rhythmic in the AVPV, however, mRNA of Dbp in the AVPV accumulated with a robust diurnal rhythm in proestrus, but not on the first day of diestrus. Thus, these results suggest that Dbp and estrogen regulate the expression of Kiss1 in the AVPV, thereby mediating the GnRH/LH surge.

Blockade of PrRP attenuates MPTP-induced toxicity in mice.

Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of the orphan G-protein coupled receptor hGR3. PrRP has been shown to be involved in the regulation of food intake, stress responses, prolactin secretion and release, blood pressure, and the opioid system. Here we report that PrRP and its receptor, GPR10, were found in the mouse substantia nigra pars compacta (SNpc), the main location of dopaminergic (DA) neurons of the nigrostriatal system. We generated PrRP knockout (KO) mice, and then treated PrRP KO mice and their wild type (WT) littermates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neuron toxin that selectively damages DA neurons in the SNpc. We found that PrRP KO mice were resistant to MPTP-induced lesions of the nigrostriatal system. These effects were further confirmed by the intracerebroventricular injection of P2L-1C, a monoclonal antibody against PrRP into mice. Taken together, our data established a critical role of PrRP in MPTP intoxication in mice.

Cgr11 encodes a secretory protein involved in cell adhesion.

We performed comparative proteomic analyses of pituitary tumor-derived cell lines, and found a new protein, preliminarily called hydrophobestin, which was produced only in somatotrophic cells, MtT/S, but not in non-hormone-producing cells, MtT/E. Hydrophobestin is encoded by the cell growth regulatory gene, Cgr11, which is known to have growth-suppressive potential in several cell lines. We have now sought to investigate the underlying events responsible for cell growth inhibition by hydrophobestin. Immunocytochemisty revealed that hydrophobestin is localized in the Golgi apparatus of MtT/S cells and Cgr11-transfected MtT/E cells. The apparent molecular mass of the protein was determined by Westerm blot analysis of conditioned culture medium of MtT/S cells. Our data show that hydrophobestin is a secretory protein localized in the pituitary gland, adrenal gland, digestive tract, reproductive organs, and kidney. We also found that hydrophobestin promotes compact monolayer cell aggregates in PC12 cells transfected with Cgr11, however, non-transfected, vector- or EF-hand motif-deleted (DeltaEF) Cgr11-transfected PC12 cells cannot form compact cell colonies. An antibody recognizing EF-hand motifs showed strong staining in the intercellular space of both Cgr11-transfected PC12 cells and MtT/S cells (Cgr11-expressing cells). Our data suggest that hydrophobestin-mediated cell adhesion may regulate cell growth through compact cell attachment.

Estrogen suppresses the stress response of prolactin-releasing peptide-producing cells.

Prolactin-releasing peptide (PrRP) is known to be produced in A1/A2 noradrenergic neurons and to mediate the stress response. Our preliminary experiment showed that PrRP neurons in the A2 region differed between males and females in terms of c-Fos expression. In addition it has been reported that estrogen receptor alpha is detectable in A2 PrRP neurons. Therefore, we speculated that the stress response of PrRP neurons is modified by estrogen. We, therefore, examined c-Fos expression in A2 PrRP neurons during the estrous cycle and found that c-Fos accumulation in PrRP neurons was significantly decreased in estrus compared with in proestrus, metestrus and diestrus. This suggests that estrogen suppresses the activation of PrRP neurons. We thus administered diethylstilbestrol (DES) to ovariectomized rats and then added restraint stress. The data clearly showed that PrRP cells in DES-administered rats significantly suppressed c-Fos accumulation induced by stress.

Identification of autotaxin as a neurite retraction-inducing factor of PC12 cells in cerebrospinal fluid and its possible sources.

Abstract Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long-acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long-acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso-PLD). Although the lyso-PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso-PLD activity and an approximately 120-kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso-PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso-PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.