For many plasma membrane receptors including

For many plasma membrane receptors including GPCRs, their density on the cell-surface is finely controlled by various transcriptional, post-transcriptional and post-translational mechanisms, and is often a determinant of overall receptor function in a cell. To date, the transcriptional regulation and molecular basis for tissue and cell type-specific expression of GHSR have not yet been fully elucidated. There have been relatively few studies, in a limited number of species including human, chicken and fish (Kaji et al., 1998, Petersenn et al., 2001, Tanaka et al., 2003, Yeung et al., 2004), characterizing the 5′-flanking region of the GHSR gene. Previous studies of the human GHSR gene have shown that it contains a TATA-less promoter, similar to most other GPCR promoters, and suggested an alternative splicing in the 5′-untranslated (5′-UT) regions (Kaji et al., 1998, Petersenn et al., 2001). These studies also demonstrated that the minimal promoter is mapped to within a relatively small segment (∼300-bp) of the 5′-flanking region of GHSR. In addition, sequence comparison of human and fish GHSR promoters identified putative Org 25543 hydrochloride for shared transcription factors, such as AP-1, NF-1, Oct-1 and USF (Yeung et al., 2004); however, the functional significance of these transcription factors in transcriptional regulation awaits further investigation. Herein, we report data on the structural and functional characterization of the 5′-flanking region of the rat Ghsr gene. In addition, we demonstrated a highly restricted expression of Ghsr1a among endocrine cell lines of rodent origin, and replicated previous findings that original and purified RC-4B/C rat pituitary tumor cell lines express a high level of functional Ghsr1a (Falls et al., 2006, Perdonà et al., 2011). Furthermore, using newly established RC-4B/C subclones with either a high or a low level of Ghsr1a mRNA, we investigated cell type-specific expression and transcriptional regulation of Ghsr1a. Our results indicate that epigenetic changes through DNA methylation and chromatin/histone modifications make significant contributions to determining the level of Ghsr1a transcription.
Materials and methods
Results
Discussion The ghrelin–GHSR1A signaling system is a pivotal regulator of various physiological and pathological processes. Therefore, it represents one of the most promising targets for the development of new drugs particularly for the treatment of metabolic diseases such as obesity, diabetes and cancer cachexia (Kojima and Kangawa, 2005, Chen et al., 2009). A better understanding of the molecular mechanisms underlying the cell type-specific expression of GHSR1A will not only provide insights into the mechanisms of gene regulation, but may also be useful for the development of new therapeutic strategies. To this end, we first performed gene expression analyses and confirmed that substantial levels of Ghsr1a mRNA were detectable in only one cell line of endocrine origin, RC-4B/C, out of nine rodent cell lines examined, data supporting the observation of Falls et al. (2006). Subsequently, we established RC-4B/C-derived subclones (RC-4B/C-H1 and H2), expressing a high level of endogenous Ghsr1a mRNA, and demonstrated that they do indeed possess a functional receptor capable of responding to exogenous administration of ghrelin, but not des-acyl ghrelin. We believed these subclones to be valuable tools for studying Ghsr gene regulation in vitro, and thus used them for all subsequent investigations. In the present study, we also characterized the upstream structure and regulation of the rat Ghsr gene. 5′-RACE using pituitary total RNA revealed a novel 5′-UT first exon characterized by multiple TSSs mapping within a TATA-less, CpG island promoter. A comparison with the transcriptional activity of a series of deletion promoter constructs in RC-4B/C-H1 cells indicated that the minimal promoter region required for basal activity resided downstream from position −202 relative to the TSS. Thus, our findings are consistent with previous studies on the human GHSR gene (Kaji et al., 1998, Petersenn et al., 2001), which implied the presence of an alternative 5′-UT first exon and mapped a minimal promoter within a region less than 1-kb upstream from the ATG translation initiation codon. In addition, a preliminary search using the UCSC Genome Browser identified a mouse EST sequence (GenBank Accession No. AK049671) corresponding to the sequence of the first exons in rats and humans. Thus, the 5′-flanking regions of GHSR genes seem to be very similar structurally and to be conserved among these species. Although analyses of the minimal promoter regions in different species predicted a number of transcription factor binding sites, there was no evidence of definitely conserved binding sites shared among these species. To obtain clues to the specific molecular players involved in cell type-specific GHSR1A expression, we compared the gene expression profiles of selected genes between Ghsr1a-expressing and non-expressing cell lines, but no genes, even those of pituitary hormones and pituitary-specific transcription factors, showed a clear correlation with the Ghsr1a expression level (H. Inoue, unpublished observations). Thus, the molecular mechanisms that dictate cell-specific GHSR1A gene expression remain obscure.