The orexigenic hormone ghrelin defends against depressive symptoms of chronic stress.

We found that increasing ghrelin levels, through subcutaneous injections or calorie restriction, produced anxiolytic- and antidepressant-like responses in the elevated plus maze and forced swim test. Moreover, chronic social defeat stress, a rodent model of depression, persistently increased ghrelin levels, whereas growth hormone secretagogue receptor (Ghsr) null mice showed increased deleterious effects of chronic defeat. Together, these findings demonstrate a previously unknown function for ghrelin in defending against depressive-like symptoms of chronic stress.

Colocalization of ghrelin O-acyltransferase and ghrelin in gastric mucosal cells.

Ghrelin is a peptide hormone with many known functions, including orexigenic, blood glucose-regulatory, and antidepressant actions, among others. Mature ghrelin is unique in that it is the only known naturally occurring peptide to be posttranslationally modified by O-acylation with octanoate. This acylation is required for many of ghrelin's actions, including its effects on promoting increases in food intake and body weight. GOAT (ghrelin O-acyltransferase), one of 16 members of the MBOAT family of membrane-bound O-acyltransferases, has recently been identified as the enzyme responsible for catalyzing the addition of the octanoyl group to ghrelin. Although the initial reports of GOAT have localized its encoding mRNA to tissues known to contain ghrelin, it is as yet unclear whether the octanoylation occurs within ghrelin-producing cells or in neighboring cells. Here, we have performed dual-label histochemical analysis on mouse stomach sections and quantitative PCR on mRNAs from highly enriched pools of mouse gastric ghrelin cells to demonstrate a high degree of GOAT mRNA expression within ghrelin-producing cells of the gastric oxyntic mucosa. We also demonstrate that GOAT is the only member of the MBOAT family whose expression is highly enriched within gastric ghrelin cells and whose whole body distribution mirrors that of ghrelin.

Aggressive melanoma cells escape from BMP7-mediated autocrine growth inhibition through coordinated Noggin upregulation.

Bone morphogenetic proteins (BMPs) are members of the TGF-beta superfamily responsible for mediating a diverse array of cellular functions both during embryogenesis and in adult life. Previously, we reported that upregulation of BMP7 in human melanoma correlates with tumor progression. However, melanoma cells are either inhibited by or become resistant to BMP7 as a function of tumor progression, with normal melanocytes being most susceptible. Herein, real-time quantitative reverse transcriptase-polymerase chain reactions and western blotting revealed that the expression of BMP antagonist, Noggin, correlates with resistance to BMP7 in advanced melanoma cells. To test the hypothesis that coordinated upregulation of Noggin protects advanced melanoma cells from autocrine inhibition by BMP7, functional expression of Noggin in susceptible melanoma cells was achieved by adenoviral gene transfer. The Noggin-overexpressing cells exhibited a growth advantage in response to subsequent BMP7 transduction in vitro under anchorage-dependent and -independent conditions, in three-dimensional skin reconstructs, as well as in vivo in severe combined immunodeficient mice. In concordance, Noggin knockdown by lentiviral shRNA confers sensitivity to BMP7-induced growth inhibition in advanced melanoma cells. Our findings suggest that, like TGF-beta, BMP7 acts as an autocrine growth inhibitor in melanocytic cells, and that advanced melanoma cells may escape from BMP7-induced inhibition through concomitant aberrant expression of Noggin.

Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain.

The hormones leptin and ghrelin act in apposition to one another in the regulation of body weight homeostasis. Interestingly, both leptin receptor expression and ghrelin receptor expression have been observed within many of the same nuclei of the central nervous system (CNS), suggesting that these hormones may act on a common population of neurons to produce changes in food intake and energy expenditure. In the present study we explored the extent of this putative direct leptin and ghrelin interaction in the CNS and addressed the question of whether a loss of ghrelin signaling would affect sensitivity to leptin. Using histological mapping of leptin receptor and ghrelin receptor expression, we found that cells containing both leptin receptors and ghrelin receptors are mainly located in the medial part of the hypothalamic arcuate nucleus. In contrast, coexpression was much less extensive elsewhere in the brain. To assess the functional consequences of this observed receptor distribution, we explored the effect of ghrelin receptor deletion on leptin sensitivity. In particular, the responses of ad libitum-fed, diet-induced obese and fasted mice to the anorectic actions of leptin were examined. Surprisingly, we found that deletion of the ghrelin receptor did not affect the sensitivity to exogenously administrated leptin. Thus, we conclude that ghrelin and leptin act largely on distinct neuronal populations and that ghrelin receptor deficiency does not affect sensitivity to the anorexigenic and body weight-lowering actions of leptin.

Ghrelin increases the rewarding value of high-fat diet in an orexin-dependent manner.

BACKGROUND: Ghrelin is a potent orexigenic hormone that likely impacts eating via several mechanisms. Here, we hypothesized that ghrelin can regulate extra homeostatic, hedonic aspects of eating behavior. METHODS: In the current study, we assessed the effects of different pharmacological, physiological, and genetic models of increased ghrelin and/or ghrelin-signaling blockade on two classic behavioral tests of reward behavior: conditioned place preference (CPP) and operant conditioning. RESULTS: Using both CPP and operant conditioning, we found that ghrelin enhanced the rewarding value of high-fat diet (HFD) when administered to ad lib-fed mice. Conversely, wild-type mice treated with ghrelin receptor antagonist and ghrelin receptor-null mice both failed to show CPP to HFD normally observed under calorie restriction. Interestingly, neither pharmacologic nor genetic blockade of ghrelin signaling inhibited the body weight homeostasis-related, compensatory hyperphagia associated with chronic calorie restriction. Also, ghrelin's effects on HFD reward were blocked in orexin-deficient mice and wild-type mice treated with an orexin 1 receptor antagonist. CONCLUSIONS: Our results demonstrate an obligatory role for ghrelin in certain rewarding aspects of eating that is separate from eating associated with body weight homeostasis and that requires the presence of intact orexin signaling.

Characterization of a novel ghrelin cell reporter mouse.

Ghrelin is a hormone that influences many physiological processes and behaviors, such as food intake, insulin and growth hormone release, and a coordinated response to chronic stress. However, little is known about the molecular pathways governing ghrelin release and ghrelin cell function. To better study ghrelin cell physiology, we have generated several transgenic mouse lines expressing humanized Renilla reniformis green fluorescent protein (hrGFP) under the control of the mouse ghrelin promoter. hrGFP expression was especially abundant in the gastric oxyntic mucosa, in a pattern mirroring that of ghrelin immunoreactivity and ghrelin mRNA. hrGFP expression also was observed in the duodenum, but not in the brain, pancreatic islet, or testis. In addition, we used fluorescent activated cell sorting (FACS) to collect and partially characterize highly enriched populations of gastric ghrelin cells. We suggest that these novel ghrelin-hrGFP transgenic mice will serve as useful tools to better understand ghrelin cell physiology.

TFIIA plays a role in the response to oxidative stress.

To characterize the role of the general transcription factor TFIIA in the regulation of gene expression by RNA polymerase II, we examined the transcriptional profiles of TFIIA mutants of Saccharomyces cerevisiae using DNA microarrays. Whole-genome expression profiles were determined for three different mutants with mutations in the gene coding for the small subunit of TFIIA, TOA2. Depending on the particular mutant strain, approximately 11 to 27% of the expressed genes exhibit altered message levels. A search for common motifs in the upstream regions of the pool of genes decreased in all three mutants yielded the binding site for Yap1, the transcription factor that regulates the response to oxidative stress. Consistent with a TFIIA-Yap1 connection, the TFIIA mutants are unable to grow under conditions that require the oxidative stress response. Underexpression of Yap1-regulated genes in the TFIIA mutant strains is not the result of decreased expression of Yap1 protein, since immunoblot analysis indicates similar amounts of Yap1 in the wild-type and mutant strains. In addition, intracellular localization studies indicate that both the wild-type and mutant strains localize Yap1 indistinguishably in response to oxidative stress. As such, the decrease in transcription of Yap1-dependent genes in the TFIIA mutant strains appears to reflect a compromised interaction between Yap1 and TFIIA. This hypothesis is supported by the observations that Yap1 and TFIIA interact both in vivo and in vitro. Taken together, these studies demonstrate a dependence of Yap1 on TFIIA function and highlight a new role for TFIIA in the cellular mechanism of defense against reactive oxygen species.

Oxidant-specific folding of Yap1p regulates both transcriptional activation and nuclear localization.

The yeast transcriptional regulator Yap1p is a key determinant in oxidative stress resistance. This protein is found in the cytoplasm under non-stressed conditions but rapidly accumulates in the nucleus following oxidant exposure. There it activates transcription of genes encoding antioxidants that return the redox balance of the cell to an acceptable range. Yap1p localization to the nucleus requires the oxidant-specific formation of disulfide bonds in the N-terminal cysteine-rich domain (N-CRD) and/or the C-terminal cysteine-rich domain (C-CRD). H(2)O(2) exposure triggers the formation of two interdomain disulfide bonds between the N-and C-CRDs. This dually disulfide-bonded structure has been argued to mask the nuclear export signal in the C-CRD that would otherwise prevent Yap1p nuclear accumulation. The C-CRD is required for wild-type H(2)O(2) tolerance but dispensable for resistance to diamide. The Saccharomyces cerevisiae TRX2 gene, encoding a thioredoxin protein, cannot be induced by H(2)O(2) in the presence of various mutant forms of Yap1p lacking the normally functioning C-CRD. In this work, we demonstrate that the proper folding of Yap1p in the presence of H(2)O(2) is required for recruitment of the mediator component Rox3p to the TRX2 promoter in addition to the nuclear accumulation of Yap1p during stress by this oxidant. These data demonstrate that the dually disulfide-bonded Yap1p N- and C-CRDs form a bifunctional protein domain controlling both nuclear localization and transcriptional activation.

YBP1 and its homologue YBP2/YBH1 influence oxidative-stress tolerance by nonidentical mechanisms in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the transcription factor Yap1p is a central determinant of resistance to oxidative stress. Previous work has demonstrated that Yap1p is recruited from the cytoplasm to the nucleus upon exposure to the oxidants diamide and H2O2 in a process that requires the transient covalent linkage of the glutathione peroxidase Gpx3p to Yap1p. Genetic and biochemical analyses indicate that while both oxidants trigger nuclear accumulation of Yap1p, the function and regulation of this transcription factor is different under these two different oxidative stresses. Ybp1p (Yap1p-binding protein) has recently been demonstrated to be required for Yap1p-mediated H2O2 resistance but not diamide resistance. A Ybp1p homologous protein (Ybh1p/Ybp2p) was also detected in the S. cerevisiae genome. Here we compare the actions of these two closely related proteins and provide evidence that while both factors influence H2O2 tolerance, they do so by nonidentical mechanisms. A double mutant strain lacking both YBP1 and YBH1 genes is more sensitive to H2O2 and more defective in activation of Yap1p-dependent gene expression than either single mutant. Ybp1p has a more pronounced effect on these phenotypes than does Ybh1p. Protein-protein interactions between Yap1p and Ybp1p could be detected by either the yeast two-hybrid or coimmunoprecipitation approach while neither technique could demonstrate Yap1p-Ybh1p interactions. Overexpression experiments indicated that high levels of Ybh1p but not Ybp1p could bypass the H2O2 hypersensitivity of a gpx3Delta strain. Together, these data argue that these two homologous proteins act as parallel positive regulators of H2O2 tolerance.