Gastrin-releasing peptide is a growth factor for human neuroblastomas.

Synthesis of both gastrin-releasing peptide (GRP) and messenger ribonucleic acid (mRNA) has been demonstrated in pregnant sheep, but studies in women have not been reported. Therefore, we examined the uterus and placenta of pregnant women at term for synthesis of GRP and expression of GRP receptor genes BRS-3 and GRP-R. A transcript of 0.95 kilobases, corresponding to GRP mRNA, was detected in endometrium and myometrium, but not in amnion, chorion, placenta, or nonpregnant endometrium. GRP immunoreactivity (GRP-ir) was detected in half (three of six) of the endometrial (1.23 +/- 0.04 pmol/g) and myometrial (0.73 +/- 0.04 pmol/g) samples and in some, but not all, samples of amnion (one of four subjects; 0.6 pmol/g), chorion (four of five subjects; 0.8 +/- 0.2 pmol/g), placenta (two of six subjects; 0.5 +/- 0.2 pmol/g), and amniotic fluid (four of six subjects; 59 +/- 19 fmol/mL). GRP-ir was present in the maternal circulation (44 +/- 12 fmol/mL) and was higher in plasma obtained from the umbilical artery (152 +/- 14 fmol/mL) and vein (143 +/- 24 fmol/mL). The major peak of GRP-ir in pregnant endometrial tissue was larger than GRP-(1-27), as determined by gel filtration chromatography. Minor peaks were also observed: two larger than the main form and one corresponding to GRP-(18-27). mRNA for GRP receptors GRP-R and BRS-3 was detected by semiquantitative reverse transcription-PCR. For both receptors, mRNA was higher in the pregnant endometrium than in the nonpregnant endometrium but was detected in all of the uteroplacental tissues examined. GRP-R mRNA predominated in the pregnant endometrium, whereas BRS-3 mRNA predominated in the membranes and placenta. In these tissues, PCR for BRS-3 mRNA gave rise to an additional product (approximately 50 bp larger). These studies demonstrated that a peptide larger than, but related to, GRP is synthesized in the pregnant human uterus and is secreted into the maternal and fetal circulations. The detection of mRNA for GRP-R, BRS-3, and possibly a transcript variant of BRS-3 as well as the detection of a peptide larger than, but related to, GRP suggest a novel regulatory unit in the human reproductive tract.

Organ to organ communication is important in the maintenance of normal fluid and electrolyte balance and blood pressure (BP). Neural mechanisms and gastrointestinal (GIT) hormones mediate the natriuresis of an oral sodium load. The GIT is the first organ exposed to ingested nutrients and most likely to react initially to a sodium load. Sensing the amount of ingested sodium by the GIT may be an important mechanism by which sodium balance is regulated. We have reported that gastrin secreted by G‐cells in the stomach and duodenum is involved in the regulation of sodium balance and BP. Besides gastrin, glucagon‐like peptide‐1 (GLP‐1) is another gut hormone, which may also participate in regulation of sodium balance and BP. It is well known that glucose‐induced GLP‐1 secretion is sodium‐dependent. The increase in intracellular calcium needed for GLP‐1 secretion is also due to sodium‐dependent cell excitability. However, the specific effect of varying sodium concentration on GLP‐1 secretion and its potential mechanism remain to be elucidated. This study aimed to determine the effect of varying sodium concentrations on GLP‐1 secretion and its potential mechanism. Here, we show that sodium can stimulate GLP‐1 secretion and that gastrin‐releasing peptide (GRP) enhances the high sodium‐induced increase in GLP‐1 secretion in a concentration and time‐dependent manner in L‐cells. The exposure of L‐cells to high (170 mM) Na+ increases GLP‐1 protein (produced by degradation of glucagon) or proglucagon mRNA expression, while Low (90 mM) and high (170 mM) Na+ increase NFAT5 and GRP receptor (GRPR) mRNA and protein expression. Silencing NFAT5 abrogates the GRP‐mediated enhancement of high sodium‐induced increase in GLP‐1 secretion and blocks the sodium‐induced increase in GRPR mRNA and protein expressions in L‐cells. Furthermore, NFAT5 silencing reduces the GLP‐1 protein or proglucagon expression in L‐cells exposed to high sodium concentration. NFAT5 silencing also reduces GRPR expression, using the promoter reporter assay; n=3/group for all studies. These results suggest that the high sodium‐induced NFAT5 expression increases expression of GRPR; GRP acting on the increased GRPR expression increases GLP‐1 secretion from L‐cells. These data give a new perspective on the mechanisms of GLP‐1 secretion, and the direct relationship between sodium and GLP‐1. These may contribute to better utilization of existing GLP‐1 based drugs and further GLP‐1‐based drug development.Effect of NaCl or GRP on GLP‐1 secretion in CCL‐251 L cells (human colorectal adenocarcinoma cells=GLP‐1 secreting cells).The same amount of cells were exposed to 90 mM, 145 mM, or 170 mM NaCl medium, and A, treated with varying concentrations of GRP (10, 100, 500, or 1000 nmol) for 4 h, or B, treated with 1000 nmol GRP at different times (1 h, 2 h, 3 h, or 4 h). GLP‐1 in the cell culture supernatant was measured by ELISA. The results are expressed as ng/ml. *P<0.05 vs. 90 mM with or without GRP. #P<0.05 vs. others, one‐way ANOVA, Holm‐Sidak test, n=3/treatment.Figure 1

Location and Characterization of the Human GRP Receptor Expressed by Gastrointestinal Epithelial Cells

Gastrin-releasing peptide (GRP) is a neuropeptide that targets transmembrane-type receptors. Its role in allergic rhinitis (AR) has yet to be investigated. The present study utilized the nasal mucosa of AR model mice to examine GRP and GRP receptor (GRPR) expression levels, localization, and other factors to evaluate their role in AR pathology. In vivo study in an animal model. GRP and GRPR expression levels were examined in three different AR models established in BALB/c mice. In addition, a GRPR antagonist (RC-3095) was administered to AR mice to investigate its effect. The distribution of GRPR expression on mast cells in the nasal mucosa with AR was examined. Finally, we investigated the inhibitory effect of RC-3095 on allergy symptoms induced by histamine. GRP and GRPR were highly expressed in the nasal mucosal epithelium and interstitial tissues surrounding the nasal glands in AR groups according to immunostaining. GRP and GRPR expression as determined by western blotting increased in the nasal mucosa as the degree of nasal sensitization increased. In addition, the average counts of sneezing and nasal rubbing after treatment in the AR + RC-3095 group were significantly lower than those in the AR + nasal saline group. Mast cells often colocalized with GRPR around nasal glands. Moreover, RC-3095 was effective in reducing sneezing induced by histamine. The GRP-GRPR system is likely to be involved in allergic inflammation. This system may represent a novel therapeutic target for refractory AR. NA. Laryngoscope, E377-E384, 2018.

Gastrin-releasing peptide (GRP) belongs to the family of bombesin-like peptides. GRP was demonstrated to stimulate the proliferation and invasiveness of androgen-independent prostate carcinoma. GRP mediates its action through the membrane-bound receptor, GRP receptor (GRPR), which is characterized by a high-affinity binding for both GRP and bombesin. In human prostate cancer tissue, GRPR mRNA was reported to be detectable in more than 90% but its immunolocalizaition has not been reported. Therefore, in this study we immunolocalized GRPR in 51 human prostate cancer cases and correlated the findings with several clinicopathological parameters in order to better understand the function and regulation of GRPR in human prostate cancer. GRPR was immnolocalized in carcinoma cells and their values were significantly associated with Gleason score and immunoreactivity of estrogen receptor βcx (ERβcx) that is one of splicing variants of ligand dependent transcription factor, ERβ, and considered to be prognostic factor of prostate cancer patients. The amounts of GRPR and ERβcx mRNA in three prostate cancer cell lines PC-3, DU-145 and LNCaP evaluated by quantitative RT-PCR (qPCR) analysis were also significantly correlated. In addition, we established stable transformants of prostate carcinoma cell line PC-3 introduced with ERβcx, and confirmed that GRPR mRNA was induced in ERβcx over-expressing PC-3 cells by qPCR analysis. These results also suggest that ERβcx contributes to prostate cancer development possibly through mediating GRPR expression in carcinoma cells.

Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: Molecular basis for dual targeting of tumoral vasculature

Gastrin-Releasing Peptide-Expressing Nerves Comprise Subsets of Human Cutaneous Aδ and C Fibers that May Sense Pruritus

Activation of gastrin-releasing peptide receptor (GRPR) in human airways has been associated with a proliferative response of bronchial cells to gastrin-releasing peptide and with long-term tobacco use. The GRPR gene is located on the X chromosome and escapes X-chromosome inactivation, which occurs in females. Increasing evidence demonstrates that women are more susceptible than men to tobacco carcinogenesis. We hypothesized that the susceptibility of women to the effects of tobacco may be associated with airway expression of GRPR. We analyzed GRPR messenger RNA (mRNA) expression in lung tissues and cultured airway cells from 78 individuals (40 males and 38 females) and in lung fibroblasts exposed to nicotine in vitro. Nicotinic acetylcholine receptors in airway cells were assayed by use of radioactively labeled nicotine and nicotine antagonists. A polymorphism in exon 2 of the GRPR gene was used to detect allele-specific GRPR mRNA expression in some individuals. Statistical tests were two-sided. GRPR mRNA expression was detected in airway cells and tissues of more female than male nonsmokers (55% versus 0%) and short-term smokers (1-25 pack-years [pack-years = number of packs of cigarettes smoked per day multiplied by the number of years of smoking]) (75% versus 20%) (P =.018 for nonsmoking and short-term smoking females versus nonsmoking and short-term smoking males). Female smokers exhibited expression of GRPR mRNA at a lower mean pack-year exposure than male smokers (37.4 pack-years versus 56.3 pack-years; P =.037). Lung fibroblasts and bronchial epithelial cells exhibited high-affinity, saturable nicotinic acetylcholine-binding sites. Expression of GRPR mRNA in lung fibroblasts was elevated following exposure to nicotine. Our results suggest that the GRPR gene is expressed more frequently in women than in men in the absence of smoking and that expression of this gene is activated earlier in women in response to tobacco exposure. The presence of two expressed copies of the GRPR gene in females may be a factor in the increased susceptibility of women to tobacco-induced lung cancer.

Bombesin-like peptides (BLPs), which have been implicated in the regulation of growth of prostatic carcinoma cells, are a product of neuroendocrine cells frequently found in prostate tissue and are postulated to play a role in the initiation or progression of prostatic carcinoma. In this report, we examined the expression, in human prostate tissue, of mRNA encoding the 3 known receptors that respond to BLPs in humans, i.e., gastrin-releasing peptide (GRP) receptor, neuromedin B (NMB) receptor and bombesin receptor subtype 3 (BRS-3). Competitive rt-PCR experiments demonstrated the widespread but variable expression of GRP receptor mRNA in fresh-frozen specimens of prostatic carcinoma (12 cases) and benign prostatic hypertrophy (6 cases). NMB receptor mRNA expression was also widespread, but its level was less variable than GRP receptor message. In contrast, we could not detect BRS-3 mRNA in most tissue samples by rt-PCR. To address which cells in the prostate express the GRP receptor, we used in situ hybridization methods to stain selectively GRP receptor mRNA. GRP receptor mRNA was expressed predominantly in the luminal and basal epithelial cells in both histologically normal and cancerous glands within sections of normal (3 cases) and diseased (37 cases) tissue. GRP receptor mRNA staining in cancerous tissue ranged widely from very intense to not detectable (about 30% of the cases), while normal tissue consistently displayed a low level of message staining. Taken together, our results demonstrate expression of the GRP receptor in a high percentage of basal and/or luminal epithelial cells of normal and diseased prostate tissues.

We have previously reported that high expression of gastrin-releasing peptide receptors (GRP-R), a member of the G-protein coupled receptor family, is observed in more undifferentiated human neuroblastoma. We have also found that GRP-R-mediated cell signaling plays a critical role in neuroblastoma cell transformation; however, its molecular mechanisms are unknown. Here, we sought to determine the role of focal adhesion kinase (FAK), a non-receptor protein tyrosine kinase, in GRP-R-mediated neuroblastoma cellular transformation. METHODS. Using human neuroblastoma tissue sections, the correlation of GRP-R and FAK expression was examined. Human neuroblastoma cell line, BE(2)-C and SK-N-SH were used for our experiments. Using transfection with Lipofectamine 2000, the level of knockdown and overexpression of GRP-R and FAK were modulated. MTT assay was carried out to measure the cell viability of neuroblastoma cells, and soft agar colony formation assay was performed to determine the capacity of cell transformation. Transwell migration and wound healing assay were employed to study cell motility. Rescue experiments by overexpression of FAK in GRP-R silencing cells were conducted to further validate the role of FAK in neuroblastoma cell transformation. Using FAK inhibitor and GRP-R agonist, animal experiment was performed. RESULTS. FAK expression correlated with GRP-R expression in BE(2)-C and SK-N-SH human neuroblastoma cells. GRP treatment induced phosphorylation of FAK at Y397 in both cell lines. Overexpression of GRP-R in SK-N-SH cells, which are known to express low endogenous levels of GRP-R, increased the levels of phosphorylated and total FAK. Moreover, overexpression of FAK increased cell viability and colony formation of SK-N-SH cells. These effects were reversed when cells were co-transfected with FAK siRNA in BE(2)-C cells, which endogenously express higher levels of GRP-R. Conversely, silencing of GRP-R decreased the levels of phosphorylated and total FAK in BE(2)-C cells. Interestingly, in cells with GRP-R silencing, FAK overexpression significantly enhanced cell viability and colony formation. GRP-R-mediated liver metastasis suppressed by FAK inhibitor in vivo. CONCLUSIONS. Our results suggest that FAK is a critical downstream regulator of GRP-R-mediated tumorigenesis. Furthermore, GRP-R and FAK expression correlate with morphological changes implicated in the malignant transformation of human neuroblastoma cells. Importantly, inhibition of FAK can suppress GRP-R-induced tumor progression in neuroblastoma in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 169. doi:1538-7445.AM2012-169

Bombesin (BN) or its mammalian counterpart, Gastrin-releasing peptide (GRP) is produced by pulmonary neuroendocrine cells (PNEC). The function of GRP as a growth factor involves lung morphogenesis, but the precise mechanism and the target cells have not been defined. We used a nonradioactive in situ hybridization (NISH) and Northern blot analysis for both GRP receptor (GRP-R) and its ligand GRP on samples of fetal and newborn human and rabbit lung. For immunolocalization of BN/GRP and the proliferating lung cell population we used anti-BN/GRP or MIB-1 antibodies, respectively. NISH and Northern blot showed peak expression of GRP-R mRNA on day 24 gestation in the fetal rabbit lung. At the cellular level, GRP-R mRNA was localized mainly in the distal airway epithelial tubes and surrounding mesenchyme, whereas proximal airways showed decreased signal. Epithelium expressing GRP-R showed positive labeling for proliferation antigen in contrast to PNEC, which were negative. In human post-natal lung, strong signal for GRP-R mRNA was localized in the airway epithelial cells and submucosal glands. PNEC in both rabbit and human lung expressed mRNA for GRP, but only in human lung were they immunoreactive for the peptide. However BN/GRP immunoreactive cells were detected in rabbit fetal lung culture. The expression of GRP-R in mammalian lung is developmentally regulated, peaking both spatially and temporally during the phase of rapid airway growth and differentiation. Both epithelial and mesenchymal components express GRP-R consistent with paracrine mechanism for GRP activity during lung morphogenesis.

Expression of gastrin-releasing peptide (GRP) and GRP receptors in the pregnant human uterus at term

Aims: To establish whether gastrin releasing peptide (GRP) and the GRP receptor (GRPR) are expressed together in gastrointestinal carcinoid tumours. Methods: Twenty six carcinoid tumours from the stomach, small intestine,...

Gastrin-releasing peptide is a neuroendocrine homolog of bombesin that demonstrated important growth-stimulatory effects in various types of cancer. High levels of expression of gastrin-releasing peptide receptors (GRPR) has been found in different malignancies, and the studies exploring the therapeutic use of GRPR antagonists have shown promising results. Our aim was to determine the GRPR expression in epidermoid carcinoma of the anal canal and discuss its potential clinical applications. We performed immunohistochemical analysis for GRPR on formalin-fixed and paraffin-embedded tumor samples obtained from 35 patients with anal cancer. As a control group, we analyzed 24 samples of nonmalignant anal tissues (hemorrhoidectomy specimens). GRPR expression was evaluated using a semiquantitative approach according to the intensity and distribution of staining. All analyzed tissues, except 1 control sample, showed positive GRPR immunoexpression. GRPR was strongly expressed in 54% of cancer specimens as compared with only 12% of the control specimens (P<0.003). In tumors, the receptor showed a diffuse and homogenous pattern of distribution within the specimens. In contrast, control specimens showed a focal pattern of staining restricted to the basal half of the epithelium. In conclusion, we demonstrated that GRPR is highly expressed in epidermoid carcinoma of the anal canal, suggesting this receptor might have a role in anal carcinogenesis. Our results provide a basis for exploiting GRPR as a target for diagnostic and therapeutic purposes in the anal cancer.

Gastrin-Releasing Peptide Receptors in Non-Neoplastic and Neoplastic Human Breast