Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor-knockout mouse.

BACKGROUND & AIMS: The G-protein-coupled receptor GPR39 is a member of a family that includes the receptors for ghrelin and motilin. Recently the peptide obestatin was identified as a natural ligand for GPR39. The objective of this study was to gain insight into the biological function of the GPR39 receptor. METHODS: GPR39(-/-) mice were generated and analyzed. RESULTS: Endogenous GPR39 expression was detected in the brain (septum-amygdala) and the gastrointestinal system (parietal cells, enterocytes, neurons, and pancreas). Gastric emptying of a solid meal (measured by the (14)C octanoic breath test) in GPR39(-/-) mice was accelerated significantly with a gastric half-emptying time of 49.5 +/- 2.2 minutes compared with 86.9 +/- 8.4 minutes in GPR39(+/+) mice. A more effective expulsion of distally located pellets (30%-75% of length) was observed in the colon of GPR39(-/-) mice. Four hours after pylorus ligation, the volume of gastric secretion was increased significantly (GPR39(-/-): 638 +/- 336 microL; GPR39(+/+): 225 +/- 170 microL), but gastric acid secretion was unchanged. The mature body weight and body fat composition of GPR39(-/-) mice was significantly higher compared with GPR39(+/+) mice, but this was not related to hyperphagia because 24-hour food intake did not differ between both genotypes. In contrast, deficiency of the GPR39 receptor led to reduced hyperphagia after fasting. The cholesterol levels were increased significantly in the GPR39(-/-) mice. CONCLUSIONS: Our data partially confirm and extend the described in vivo effects of obestatin and suggest that this peptide plays a functional role in the regulation of gastrointestinal and metabolic function through interaction with the GPR39 receptor.

p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2.

The Chk2 kinase is a tumor suppressor and key component of the DNA damage checkpoint response that encompasses cell cycle arrest, apoptosis, and DNA repair. It has also been shown to have a role in replicative senescence resulting from dysfunctional telomeres. Some of these functions are at least partially exerted through activation of the p53 transcription factor. High-level expression of virally transduced Chk2 in A549 human lung carcinoma cells led to arrested proliferation, apoptosis, and senescence. These were accompanied by various molecular events, including p21(Waf1/Cip1) (p21) transcriptional induction, consistent with p53 activation. However, Chk2-dependent senescence and p21 transcriptional induction also occurred in p53-defective SK-BR-3 (breast carcinoma) and HaCaT (immortalized keratinocyte) cells. Small interfering RNA-mediated knockdown of p21 in p53-defective cells expressing Chk2 resulted in a decrease in senescent cells. These results revealed a p53-independent role for Chk2 in p21 induction and senescence that may contribute to tumor suppression and genotoxic treatment outcome.

Transcriptional response to corticotropin-releasing factor in AtT-20 cells.

Corticotropin-releasing factor (CRF) plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis, mediating endocrine and behavioral responses to various stressors. Two high-affinity receptors for CRF have been described. Although many of the intracellular signaling pathways activated by CRF have been studied extensively, our knowledge of transcriptional responses downstream of the CRF receptor 1 (CRFR1) is still limited. To elucidate gene networks regulated by CRF and CRFR1, we applied microarray technology to explore transcriptional response to CRF stimulation. Therefore, mouse pituitary-derived AtT-20 cells were exposed continuously to CRF either in the presence or absence of the specific CRFR1 antagonist R121919. Transcriptional responses to different treatments were studied in a time course ranging from 0.5 to 24 h. Microarray data were analyzed using classic microarray data analysis tools such as correspondence factor analysis, cluster analysis, and fold-change filtering. Furthermore, spectral map analysis was applied, a recently introduced unsupervised multivariate analysis method. A broad and transient transcriptional response to CRF was identified that could be blocked by the antagonist. This way, several known CRF-induced target genes and novel CRF responsive genes were identified. These include transcription factors such as cAMP-responsive element modulator (7x increased), secreted peptides such as cholecystokinin (1.5x), and proteins involved in modulating intracellular signaling, such as regulator of G-protein signaling 2 (11x). Up-regulation of many of these genes can be explained as negative feedback, attenuating CRF-activated pathways. In addition, spectral map analysis proved to be a promising new tool for microarray data analysis.

Graphical exploration of gene expression data: a comparative study of three multivariate methods.

This article describes three multivariate projection methods and compares them for their ability to identify clusters of biological samples and genes using real-life data on gene expression levels of leukemia patients. It is shown that principal component analysis (PCA) has the disadvantage that the resulting principal factors are not very informative, while correspondence factor analysis (CFA) has difficulties interpreting distances between objects. Spectral map analysis (SMA) is introduced as an alternative approach to the analysis of microarray data. Weighted SMA outperforms PCA, and is at least as powerful as CFA, in finding clusters in the samples, as well as identifying genes related to these clusters. SMA addresses the problem of data analysis in microarray experiments in a more appropriate manner than CFA, and allows more flexible weighting to the genes and samples. Proper weighting is important, since it enables less reliable data to be down-weighted and more reliable information to be emphasized.