Involvement of MCH-oxytocin neural relay within the hypothalamus in murine nursing behavior.

Multiple sequential actions, performed during parental behaviors, are essential elements of reproduction in mammalian species. We showed that neurons expressing melanin concentrating hormone (MCH) in the lateral hypothalamic area (LHA) are more active in rodents of both sexes when exhibiting parental nursing behavior. Genetic ablation of the LHA-MCH neurons impaired maternal nursing. The post-birth survival rate was lower in pups born to female mice with congenitally ablated MCH neurons under control of tet-off system, exhibiting reduced crouching behavior. Virgin female and male mice with ablated MCH neurons were less interested in pups and maternal care. Chemogenetic and optogenetic stimulation of LHA-MCH neurons induced parental nursing in virgin female and male mice. LHA-MCH GABAergic neurons project fibres to the paraventricular hypothalamic nucleus (PVN) neurons. Optogenetic stimulation of PVN induces nursing crouching behavior along with increasing plasma oxytocin levels. The hypothalamic MCH neural relays play important functional roles in parental nursing behavior in female and male mice.

Growth hormone increases regulator of calcineurin 1-4 (Rcan1-4) mRNA through c-JUN in rat liver.

Growth hormone (GH) activates multiple signal transduction pathways. To investigate these pathways, we identified novel genes whose transcription was induced by GH in the liver of hypophysectomized (HPX) rats using the suppression subtractive hybridization technique. We found that regulator of calcineurin 1 (Rcan1) mRNA was upregulated by GH administration. RCAN1 regulates the activity of calcineurin, a Ca/calmodulin-dependent phosphatase. Rcan1 encodes two major transcripts, Rcan1-1 and Rcan1-4, resulting from differential promoter use and first exon choice. We found that a single injection of GH increased the levels of Rcan1-4 mRNA and RCAN1-4 protein transiently, but did not increase Rcan1-1 mRNA in HPX rat liver. Then the molecular mechanism of GH to induce Rcan1-4 transcription was examined in rat hepatoma H4IIE cells. Experiments using inhibitors suggested that c-JUN N-terminal kinase was required for the induction of Rcan1-4 mRNA by GH. GH increased the levels of phosphorylated c-JUN protein and c-Jun mRNA in HPX rat liver. The luciferase and electrophoretic mobility shift assays showed that c-JUN upregulated Rcan1-4 mRNA by binding to the cAMP-responsive element in the upstream of Rcan1 exon 4. These results indicate that GH activates c-JUN to affect the activity of calcineurin by the induction of Rcan1-4 in rat liver.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein ß pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.

Vomeronasal signal deficiency enhances parental behavior in socially isolated male mice.

We previously reported that social isolation promotes parental care in sexually naïve male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an adjacent compartment. In the present study, we examined whether the chemosensory signals detected in the vomeronasal organ (VNO) are involved in parental behavior by using mice deficient for a VNO-specific ion channel (Trpc2-/-) and thus impaired in VNO-input signaling. We housed virgin homozygous Trpc2-/- and heterozygous Trpc2± males for 3weeks during puberty (5-8weeks old) alone or in groups of 3-5 males. At 8weeks of age, the mice were placed with three pups in an observation cage and tested for parental behavior. The Trpc2-/- males housed under isolated conditions spent significantly longer in the vicinity of pups than did the Trpc2-/- males than had been group housed, whereas no isolation effect was observed in heterozygous Trpc2± males. Both Trpc2 knockout and isolation housing significantly increased the time males spent licking pups and crouching (arched back posture over pups to enable nursing), whereas only isolation housing increased the incidence of retrieval behavior. These results demonstrated that social signals transmitted not only through the VNO but also from other modalities, independent of each other, suppress the expression of parental behavior during puberty in sexually naïve males.

Social isolation prompts maternal behavior in sexually naïve male ddN mice.

Maternal behavior in mice is considered to be sexually dimorphic; that is, females show maternal care for their offspring, whereas this behavior is rarely shown in males. Here, we examined how social isolation affects the interaction of adult male mice with pups. Three weeks of isolation during puberty (5-8 weeks old) induced retrieving and crouching when exposed to pups, while males with 1 week isolation (7-8 weeks old) also showed such maternal care, but were less responsive to pups. We also examined the effect of isolation during young adulthood (8-11 weeks old), and found an induction of maternal behavior comparable to that in younger male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an attached compartment separated by doubled opaque barriers. These results demonstrate that social isolation in both puberty and postpuberty facilitates male maternal behavior in sexually naïve mice. The results also indicate that air-borne chemicals and/or sounds of male conspecifics, including ultrasonic vocalization and noise by their movement may be sufficient to interfere with the isolation effect on induction of maternal behavior in male mice.

Sunitinib, a small-molecule receptor tyrosine kinase inhibitor, suppresses neointimal hyperplasia in balloon-injured rat carotid artery.

The migration and proliferation of vascular smooth muscle cells (VSMCs) induced by growth factors play a critical role in in-stent stenosis after percutaneous coronary intervention (PCI). The present study tested the hypothesis that sunitinib malate (sunitinib), a tyrosine kinase inhibitor of multiple receptors for growth factors, can reduce neointimal formation after arterial injury in vivo and sought to reveal the underlying mechanism in vitro. Male Wistar rats with balloon-injured carotid arteries were administered either sunitinib or a vehicle orally for 2 weeks. Sunitinib significantly inhibited neointimal hyperplasia relative to control by reducing active cell proliferation. In cultured human aortic smooth muscle cells (HASMCs), sunitinib significantly inhibited platelet-derived growth factor (PDGF)-induced increases of DNA synthesis, cell proliferation, and migration relative to controls as evaluated by [(3)H] thymidine incorporation, cell number, and the Boyden chamber assay, respectively. Immunoblot analyses showed that sunitinib suppressed phosphorylation of PDGF-BB inducible extracellular signal-regulated kinase and autophosphorylation of PDGF ß-receptor, which are the key signaling steps involved in HASMC activation. These results indicate that sunitinib inhibits neointimal formation after arterial injury by suppressing VSMC proliferation and migration presumably through inactivation of PDGF signaling. As such, it may be a potential therapeutic agent, which targets arterial restenosis after PCI.

Adiponectin inhibits macrophage tissue factor, a key trigger of thrombosis in disrupted atherosclerotic plaques.

OBJECTIVE: Adiponectin (APN) is an adipocytokine with anti-atherogenic and anti-inflammatory properties. Hypoadiponectinemia may associate with increased risk for coronary artery disease (CAD) and acute coronary syndrome (ACS). Tissue factor (TF) initiates thrombus formation and facilitates luminal occlusion after plaque rupture, a common cause of fatal ACS. This study tested the hypothesis that APN influences TF expression by macrophages (MΦ), inflammatory cells found in atheromatous plaques. METHODS: Human monocyte-derived MΦ or RAW 264.7 cells transfected with TF promoter construct, pretreated with a physiological range of recombinant APN (1-10 µg/ml), received LPS stimulation. TF mRNA and protein levels were quantified by real-time RT-PCR and ELISA. TF pro-coagulant activity was evaluated by one-step clotting assay. TF promoter activity was determined by a dual-luciferase reporter assay. Immunoblot analyses assessed intracellular signaling pathways. RESULTS: APN treatment suppressed TF mRNA expression and protein production in LPS-stimulated human MΦ, compared to vehicle controls. APN treatment also significantly reduced TF pro-coagulant activity in lysates of LPS-stimulated human MΦ, compared to vehicle controls. Moreover, APN suppressed TF promoter activity in LPS-stimulated MΦ compared to controls. APN suppressed phosphorylation and degradation of IκB-α in LPS-stimulated MΦ. CONCLUSIONS: APN reduces thrombogenic potential of MΦ by inhibiting TF expression and activity. These observations provide a potential mechanistic link between low APN levels and the thrombotic complications of atherosclerosis.

Melanocortin 2 receptor is required for adrenal gland development, steroidogenesis, and neonatal gluconeogenesis.

ACTH (i.e., corticotropin) is the principal regulator of the hypothalamus-pituitary-adrenal axis and stimulates steroidogenesis in the adrenal gland via the specific cell-surface melanocortin 2 receptor (MC2R). Here, we generated mice with an inactivation mutation of the MC2R gene to elucidate the roles of MC2R in adrenal development, steroidogenesis, and carbohydrate metabolism. These mice, the last of the knockout (KO) mice to be generated for melanocortin family receptors, provide the opportunity to compare the phenotype of proopiomelanocortin KO mice with that of MC1R-MC5R KO mice. We found that the MC2R KO mutation led to neonatal lethality in three-quarters of the mice, possibly as a result of hypoglycemia. Those surviving to adulthood exhibited macroscopically detectable adrenal glands with markedly atrophied zona fasciculata, whereas the zona glomerulosa and the medulla remained fairly intact. Mutations of MC2R have been reported to be responsible for 25% of familial glucocorticoid deficiency (FGD) cases. Adult MC2R KO mice resembled FGD patients in several aspects, such as undetectable levels of corticosterone despite high levels of ACTH, unresponsiveness to ACTH, and hypoglycemia after prolonged (36 h) fasting. However, MC2R KO mice differ from patients with MC2R-null mutations in several aspects, such as low aldosterone levels and unaltered body length. These results indicate that MC2R is required for postnatal adrenal development and adrenal steroidogenesis and that MC2R KO mice provide a useful animal model by which to study FGD.

Regulation of corticotropin-releasing factor (CRF) type-1 receptor gene expression by CRF in the hypothalamus.

We reported previously that acute stress and intracerebroventricular (i.c.v.) injection of corticotropin-releasing factor (CRF) increased neuronal activation and CRF type-1 receptor (CRFR-1) mRNA expression in the CRF-producing neurons of the parvocellular paraventricular nucleus (PVN) of the hypothalamus. In this study, to determine whether CRF can act directly on hypothalamic CRF neurons, thereby increasing CRFR-1 expression, microinjection of CRF into PVN neurons in vivo and primary cultures of dispersed rat fetal hypothalami in vitro were performed. Microinjection of 0.1 microg of CRF into the PVN significantly increased c-fos and CRFR-1 mRNA expression in the CRF-producing parvocellular PVN, 30 min or 180 min after injection, respectively. This effect was blocked by a CRF antagonist, alpha-helical CRF. CRF, when injected into the lateral ventricle at the same dose, increased neither CRFR-1 nor c-fos mRNA levels in the PVN. Primary culture of hypothalamic neurons revealed that CRFR-1 like immunoreactivity was located in CRF-containing neurons, and that the CRFR-1 mRNA level was significantly increased 4 h after incubation with 10(-8) M CRF. These results demonstrate that CRF directly affects hypothalamic neurons to increase CRFR-1 mRNA expression, providing evidence of a direct role for CRF in the regulation of CRFR-1 expression of hypothalamic neurons.