Identification of Physiologically Active Substances as Novel Ligands for MRGPRD

Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent

MRGD, a MAS-related G-protein Coupled Receptor, Promotes Tumorigenisis and Is Highly Expressed in Lung Cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target

Gene expression profiling of normal human fibroblasts treated by ionizing radiation

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IHC analysis of MRGD expression.

<p>Cell block samples were used to confirm the antibody specificity in immunostaining. HEK293/αvβ3 cells transfected with MRGD expression vector (<b>A</b>) or Mock vector (<b>B</b>) are shown. <b>C.</b> The representative staining of lung adenocarcinoma which is positive for MRGD.</p

Tumorigenesis of NIH3T3 cells stably expressing MRGD (NIH3T3-MRGD cells).

<p><b>A.</b> Representative pictures of focus formation in monolayer cultures of NIH3T3 cells stably expressing Mock (NIH3T3-Mock cells, left) or MRGD (NIH3T3-MRGD cells, right). Cells were stained with crystal violet after fixing with 4% paraformaldehyde. <b>B.</b> Representative pictures of NIH3T3-MRGD or NIH3T3-Mock spheroid on Days 1 and 7. <b>C.</b> Spheroid growth curves. The spheroid sizes of NIH3T3-MRGD (closed) or NIH3T3-Mock (open) at Days 2, 3, 5 and 7, are shown with their diameters (mean ± SD). * indicates p<0.005 (Mann-Whitney U test, 2 tails). <b>D.</b> Cell proliferation of spheroid cultures of NIH3T3-MRGD or NIH3T3-Mock. Luminescence of whole cell ATP contents (means ± SD) was measured at 6 days after plating. * indicates p<0.005 (Mann-Whitney U test, 2 tails).</p

MRGD ligand, beta-alanine, promoted cell proliferation of NIH3T3-MRGD cells.

<p>NIH3T3 cells transfected with MRGD or RASV12 were cultured in RPMI1640 containing 0.1% BSA and various concentration of beta-alanine for 7 days. Data were obtained from three independent cell cultures (means ± SD). * indicates p<0.05 (Mann-Whitney U test, 2 tails), compared with NIH3T3-MRGD without beta-alanine, and with NIH3T3-RASV12.</p

Tumor volumes of NIH3T3-MRGD or Mock bearing mice.

<p>Tumor volumes (mm³) were calculated according to the following equations: Tumor volume (mm³) = 1/2×(tumor length)×(tumor width)²</p

Quantitative RT-PCR analysis of MRGD mRNA expression in clinical samples.

<p>One hundred and twenty seven sets of RNA samples of cancer and non-cancer portions from the same patients with lung (n = 33), esophagus (n = 12), breast (n = 16), kidney (n = 10), stomach (n = 25), uterus (n = 12) or colon (n = 19) cancer. Ratios of the amount of MRGD mRNA in tumor portion per that in normal portion of each case were plotted. The bar indicates the mean of the ratio in each cancer type.</p