Abstract
The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.
Highlights
Ghrelin is a multifunctional 28-amino acid hormone mainly produced in the stomach [1], and produced by a wide variety of tissues where it can act as a paracrine/autocrine factor [2]
Results of the current study confirm, as indicated previously in Gahete et al [35], and, for the first time demonstrate, that the human ghrelin gene, similar to that reported in the mouse gene [26], can retain In1, one type of alternative splicing likely associated with the failure of intron detection which occurs more frequently in short introns flanked by weak splice sites [29]
The first 36aa of the prepro-In1-ghrelin, including the signal peptide, and the first 12aa of the mature native-ghrelin, including the putative acylation site at Ser3 and the residues found to be necessary for acylation (Gly1 and Phe4) [4], would be identical to that encoded by the native-prepro-ghrelin mRNA
Summary
Ghrelin is a multifunctional 28-amino acid (aa) hormone mainly produced in the stomach [1], and produced by a wide variety of tissues where it can act as a paracrine/autocrine factor [2]. The acyl-ghrelin/GHSR1a system has been directly associated with multiple physiological functions related to regulation of energy balance and metabolic function at the central and peripheral level [5,6]. Ghrelin and its receptor are present in many endocrine and non-endocrine tumor cell types (for example, gastroenteropancreatic, pituitary, prostate, breast), and in their related cancer cell lines, where ghrelin has been shown to control neoplastic cell proliferation [7,8,9,10]. A truncated isoform of GHSR1a, the GHSR type 1b (GHSR1b), is found in the majority of the tumors and cancer cell lines cited above, its potential role in tumor regulation remains unknown [11,12]. There is only isolated evidence that GHSR1b can act as a co-receptor with the neurotensin receptor 1 to form a novel receptor for neuromedin U in lung cancer [13]
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