G-protein coupled receptor agonists mediate Neu1 sialidase and matrix metalloproteinase-9 cross-talk to induce transactivation of TOLL-like receptors and cellular signaling

The mechanism(s) behind GPCR transactivation of TLR receptors independent of TLR ligands is unknown. Here, GPCR agonists bombesin, bradykinin, lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and -2, but not thrombin induce Neu1 activity in live macrophage cell lines and primary bone marrow macrophage cells from wild-type (WT) mice but not from Neu1-deficient mice. Using immunocytochemistry and NFκB-dependent secretory alkaline phosphatase (SEAP) analyses, bombesin induced NFκB activation in BMC-2 and RAW-blue macrophage cells, which was inhibited by MyD88 homodimerization inhibitor, Tamiflu, galardin, piperazine and anti-MMP-9 antibody. Bombesin receptor, neuromedin B (NMBR), forms a complex with TLR4 and MMP9. Silencing MMP9 mRNA using siRNA transfection of RAW-blue macrophage cells markedly reduced Neu1 activity associated with bombesin-, bradykinin- and LPA-treated cells to the untreated controls. These findings uncover a molecular organizational GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of TLR receptors and subsequent cellular signaling.     

Proadrenomedullin N-terminal 20 peptide (PAMP) elevates blood glucose levels via bombesin receptor in mice

We found a potent hyperglycemic effect of proadrenomedullin N-terminal 20 peptide (PAMP) after intra-third cerebroventricular administration at a dose of 10 nmol in fasted mice. PAMP has four homologous residues with bombesin (BN), a hyperglycemic peptide. PAMP showed affinity for gastrin-releasing peptide preferring receptor (GRP-R) and neuromedin B preferring receptor. The PAMP-induced hyperglycemic effect was inhibited by [D-Phe(6), Leu-NHEt(13), des-Met(14)]-BN (6-14), GRP-R specific antagonist, indicating that the hyperglycemic effect is mediated at least in part via GRP-R. Furthermore, pretreatment of alpha-adrenergic blocker inhibited the PAMP-induced hyperglycemia and hyperglucagonemia, suggesting that the increase of glucagon secretion through alpha-adrenergic activation is involved in this hyperglycemic effect of PAMP.     

Structure and chromosomal localization of the mouse bombesin receptor subtype 3 gene

Bombesin (BN)-like peptides/neurotransmitters mediate a broad range of physiological funtions in the gastrointestinal tract and the central nervous system through binding to their specific, high-affinity mammalian bombesin receptors. This family of heptahelical, G protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R, or bb2), neuromedin B receptor (NMB-R, or bb1), and the bombesin receptor subtype 3 (BRS-3, or bb3). The tissue distribution of BRS-3 is quite dissimilar compared to the other two BN receptors, GRP-R and NMB-R, and a natural ligand for BRS-3 is currently unknown. Nothing is known about mechanisms regulating BRS-3 gene expression and possible association with disease. To gain insight into the underlying structure and chromosomal localization of the BRS-3 genes, bacteriophage P1 genomic clones, harboring the genes for the human and mouse BRS-3, respectively, were isolated and their structure and chromosomal localizations determined. The protein-coding region of both genes is divided into three exons and spans approximately 5 kb. The loci of the BRS-3 genes were mapped to a syntenic region of the human (Xq25) and mouse (XA7.1–7.2) X-chromosome, respectively. The structural data of the BRS-3 genes derived from this study will permit future investigations of the mechanisms regulating their expression.     

The bombesin receptor subtypes have distinct G protein specificities

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Galphaq and mouse Galphaq but not bovine retinal Galphat or bovine brain Galphai/o. The GRP-R- and NMB-R-catalyzed activations of Galphaq were dependent upon and enhanced by different betagamma dimers in the same rank order as follows: bovine brain betagamma > beta1gamma2 > beta1gamma1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain betagamma, beta1gamma1, and beta1gamma2 and squid retinal Galphaq. In addition, GRP-R showed higher catalytic activity on squid Galphaq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgammaS binding to Galphai/o or Galphat. However, BRS-3 showed little, if any, coupling with squid Galphaq but clearly activated mouse Galphaq. GRP-R and NMB-R catalyzed GTPgammaS binding to both squid and mouse Galphaq, with GRP-R activating squid Galphaq more effectively, and NMB-R also showed slight preference for squid Galphaq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galphaq family.     

Development and function of bombesin-like peptides and their receptors

Amphibian bombesin and its related peptides consist a family of neuropeptides in many vertebrate species. Bombesin and two major bombesin-like peptide in mammals, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to elicit various physiological effects. These include inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. Receptors for GRP and NMB (GRP-R and NMB-R), as well as third subtype of bombesin-like peptide receptor (BRS-3) have been cloned. These receptors are G-protein-coupled receptors and are expressed in various brain regions and in the digestive tract. In this paper, we will summarize studies on these peptides and their receptors, with special reference to research using gene-knockout mice. These studies clearly demonstrated the role of three receptors in vivo and in vitro. We will also discuss the phylogeny of these receptors.     

Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells.

Bombesin-like peptides have been implicated as autocrine growth factors influencing the pathogenesis and progression of some human lung carcinoma cells. To determine the pharmacologic and structural properties of the bombesin receptors expressed in human lung carcinoma cells, cDNA clones encoding a human gastrin-releasing peptide receptor (GRP-R) and a pharmacologically distinct neuromedin-B preferring bombesin-receptor (NMB-R) were isolated from a human small cell lung carcinoma cell line (NCI-H345). After expression in Xenopus oocytes, a GRP-R-specific antagonist was effective in blocking responses elicited from the cloned GRP-R, but not the NMB-R. Both GRP-R and NMB-R mRNA expression was detected at varying levels in a panel of human lung cancer cell lines. These results indicate heterogeneity of bombesin receptor subtypes exists in human lung carcinoma cells and should be considered in the design of bombesin receptor antagonists intended to inhibit tumor cell growth.     

Bombesin-like peptide receptor gene expression,regulation, and function in fetal murine lung

Bombesin-peptide (BLP) immunoreactivity occurs at high levels in fetal lung. Previous studies showed that bombesin promotes fetal lung development. To test the hypothesis that such effects are mediated by known mammalian bombesin receptors [gastrin-releasing peptide (GRP)/bombesin-preferring receptor (GRPR), neuromedin B (NMB) receptor (NMBR), and the orphan bombesin receptor subtype-3 (BRS-3)], we analyzed the ontogeny of GRPR, NMBR, and BRS-3 gene expression in mouse lung. We examined the regulation of these three genes by dexamethasone and bombesin, which modulate lung development. Using incorporation of [3H]thymidine and [3H]choline, we then assessed whether GRP, NMB, and Leu8-phyllolitorin modulate lung growth and maturation in fetal lung explants. GRPR gene expression was detected predominantly in utero, whereas NMBR and BRS-3 genes were expressed from embryonic days 13-16 and on multiple postnatal days. All three mRNAs are present in airway epithelium and mesenchymal cells but occur in different relative patterns. These genes were regulated differently. Dexamethasone and bombesin increased GRPR mRNA, bombesin downregulated NMBR, and neither agent affected BRS-3. GRP increased incorporation of [3H]thymidine and [3H]choline in explants, whereas NMB induced cell proliferation and Leu8-phyllolitorin yielded variable results. Cumulative data suggest the involvement of multiple BLP receptors, including novel molecules, and argue against simple functional redundancy within this gene family during lung development.     

cDNA cloning, characterization, and brain region-specific expression of a neuromedin-B-preferring bombesin receptor.

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.     

Nitric oxide triggers enhanced induction of vascular endothelial growth factor expression in cultured keratinocytes (HaCaT) and during cutaneous wound repair.

Recently, we demonstrated a large induction of inducible nitric oxide synthase (iNOS) during cutaneous wound repair. In this study, we investigated the role of nitric oxide (NO) for the expression of vascular endothelial growth factor (VEGF), which represents the most important angiogenic factor during the proliferative phase of skin repair. Since keratinocytes are the major source of VEGF production during this process, we used cultured keratinocytes (HaCaT cell line) as an in vitro model to investigate NO action on growth factor- and cytokine-stimulated VEGF expression. Exogenously added NO enhanced transforming growth factor-beta1-, keratinocyte growth factor-, interleukin-1beta-, tumor necrosis factor-alpha-, and interferon-gamma-induced VEGF mRNA and protein synthesis in keratinocytes. We could demonstrate that high-level expression of cytokine-induced VEGF mRNA in keratinocytes is dependent on endogenously produced NO, as inhibition of the coinduced iNOS by N(G)-monomethyl-L-arginine (L-NMMA) markedly decreased cytokine-triggered VEGF mRNA levels in the cells. We also established an in vivo model in mice to investigate the role of NO during wound healing. During excisional wound repair, mice were treated with L-N(6)-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of iNOS enzymatic activity. Compared to control mice, L-NIL-treated animals were characterized by markedly reduced VEGF mRNA levels during the inflammatory phase of repair. Immunohistochemistry demonstrated reduced VEGF protein expression and a completely disorganized pattern of VEGF-expressing keratinocytes within the hyperproliferative epithelium at the wound edge in L-NIL-treated mice. We demonstrate that triggering of VEGF expression is a crucial molecular mechanism underlying NO function during wound healing.     

Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells

Bombesin and the C-terminal portion of gastrin-releasing peptide (GRP14-27) each increase clonal growth rate and colony-forming efficiency of normal human bronchial epithelial cells. These effects occur in the presence or absence of an optimal concentration (5 ng/ml) of epidermal growth factor (EGF). In contrast to EGF bombesin and GRP14-27 do not stimulate cell migration. Thus, bombesin and the C-terminal fragment of gastrin-releasing peptide represent a new class of peptides mitogenic for normal human epithelial cells.     

Activation of extracellular signal-regulated kinase mediates bombesin-induced mitogenic responses in prostate cancer cells

Bombesin and its mammalian homologue gastrin-releasing peptide have been shown to be highly expressed and secreted by neuroendocrine cells in prostate cancer, and are thought to be related to the carcinogenesis and progression of this disease. We found, in this study, bombesin specifically induced mitogen-activated protein (MAP) kinase activation as shown by increased extracellular regulated kinase (ERK) phosphorylation and epidermal growth factor (EGF) receptor transactivation in prostate cancer cells, which express functional gastrin-releasing peptide receptor. The transactivation of EGF receptor was required for bombesin-induced ERK phosphorylation. Furthermore, non-receptor tyrosine kinase Src and cellular Ca2+ were shown to be involved in bombesin-induced EGF receptor transactivation and ERK phosphorylation. Inhibition of either EGF receptor transactivation or ERK activation blocked bombesin-induced DNA synthesis in these cells. Taken together, these data suggest bombesin may act as a mitogen in prostate cancer by activating MAP kinase pathway via EGFR transactivation.     

Bombesin-like peptide and receptors in lung injury models: diverse gene expression, similar function

We previously demonstrated that bombesin-like peptide (BLP) mediates lung injury in premature infants with bronchopulmonary dysplasia (BPD). We now investigate gene expression and function of BLP (gastrin-releasing peptide, GRP) and BLP-receptors (GRP-R and BRS-3) in lung from two baboon BPD models. In the "interrupted gestation model," only GRP mRNA was up-regulated. In the "hyperoxic model," GRP-R mRNA was up-regulated. In lung explants from O2-treated animals, all BPD animals responded to 1nM bombesin, whereas non-BPD animals did not; the opposite effect was observed with a BLP blocking antibody. Cumulatively, these observations suggest that novel BLPs and/or BLP receptors are likely to be implicated in the pathogenesis of BPD.     

Biphasic regulation of HMG-CoA reductase expression and activity during wound healing and its functional role in the control of keratinocyte angiogenic and proliferative responses

In this study, we determined the regulation and potential function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) during skin repair in mice. Upon skin injury, healthy mice exhibited a biphasic increase in HMGR expression and activity with elevated levels at days 3 and 13 post-wounding. In situ hybridization revealed wound margin keratinocytes as a cellular source of HMGR expression. In vitro experiments using cultured HaCaT keratinocytes uncovered epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and insulin as potent co-inducers of HMGR activity and vascular endothelial growth factor (VEGF) in the cells. Insulin-, but not EGF-mediated VEGF protein expression was functionally connected to co-induced HMGR activity, as simvastatin restrictively interfered only with insulin-induced translation of VEGF mRNA by inhibition of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation. Functional ablation of insulin-induced sterol regulatory element-binding protein (SREBP)-2 by siRNA abolished HMGR expression and insulin-triggered VEGF protein release from keratinocytes. Simvastatin also blocked proliferation of cultured keratinocytes. The observed inhibitory effects of simvastatin on keratinocyte VEGF expression and proliferation could be reversed by mevalonate, the product of HMGR enzymatic activity. In accordance, simvastatin-mediated inhibition of HMGR activity in acutely regenerating tissue of wounded mice was paralleled by a marked loss of VEGF protein expression and disturbances of normal proliferation processes in wound margin keratinocytes during skin repair.     

Effect of bombesin on feeding behavior

Peripherally-administered bombesin and gastrin-releasing peptide produce potent, dose-related, and specific reductions of food intake at test meals in rats. Similar effects on meal size are observed after intraperitoneal injections in mice and after intravenous infusions in baboons and humans. The mechanism for this effect is unknown, but the action of bombesin is not blocked by complete subdiaphragmatic vagotomy, by a variety of peripheral endocrine and neural ablations, or by lesions of the area postrema or hypothalamus. Hypothalamic injections of bombesin produce small but specific reductions of food intake; the relationship of this central effect to the peripheral effect of the peptide is unknown. Bombesin and bombesin-like peptides may play roles in the regulation of meal size.     

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.     

Wound healing mediator production by human dermal fibroblasts grown within a collagen-GAG matrix for skin repair in humans

Cell and tissue therapy applications in humans are being used increasingly, particularly for tissue repair. Several reconstructed skin models have been proposed. Wound healing involves overlapping steps of inflammation, cell migration and proliferation, neovascularisation, extracellular matrix production and remodelling. This is regulated by numerous cytokines and other soluble mediators. We have prepared dermal substitutes (DS) consisting of a collagen-GAG, three-dimensional matrix colonized by human dermal fibroblasts (HDF), isolated by skin explant or enzymatic digestion of the skin for potential therapeutic use in humans. To test the functionality of these DS, we measured (ELISA) the stimulatory effect on HDF in the matrix, of serial dilutions of human serum (HS) on the production of wound healing mediators: interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and tissue inhibitor of metalloproteinase-1 (TIMP-1). We observed: 1). a stimulatory effect of HS on HDF production of the different mediators tested, with a dose-dependent effect in the case of IL-8 and VEGF. 2). A matrix-potentiating effect on the production of the different mediators by HDF. 3). A decrease in the production of IL-8 and VEGF when HDF isolated by enzymatic digestion was used to colonize the matrix as compared with HDF isolated by skin explant. We conclude: 1). that the production by HDF, in a collagen-GAG matrix, of mediators involved in cutaneous wound healing is decreased when HDF are isolated by enzymatic skin digestion rather than by skin explant. 2). That measurement of the production of cytokines or other mediators could be a useful quality control to test the functionality of tissue-engineered DS for tissue repair therapy in humans and more generally of cells prepared for cell therapy.     

Bombesin induces a reduction of somatostatin inhibition of adenylyl cyclase activity, Gi function, and somatostatin receptors in rat exocrine pancreas.

To analyze the effect of bombesin on the somatostatin (SS) mechanism of action in the exocrine pancreas, male Wistar rats (250-270 g) were injected intraperitoneally with bombesin (10 microg/kg) three times daily at 8-h intervals for 7 or 14 days. Bombesin attenuated the ability of SS to inhibit forskolin-stimulated adenylyl cyclase activity in pancreatic acinar membranes. However, it did not decrease the ability of forskolin to stimulate the adenylyl cyclase catalytic subunit. The ability of 5'-guanylylimidodiphosphate [Gpp(NH)p] (a nonhydrolyzable GTP analog) to inhibit forskolin-stimulated adenylyl cyclase activity was diminished in pancreatic acinar cell membranes from bombesin-treated rats. Bombesin administration did not affect the ADP-ribosylation of a 41-kDa G protein catalyzed by pertussis toxin. The maximal SS binding capacity of pancreatic acinar membranes from bombesin-treated rats was decreased when compared with controls at the two time periods studied. The bombesin/gastrin-releasing peptide antagonist [D-Tpi6,Leu13psi(CH2NH)Leu14]bombesin (6-14) (RC-3095) (10 microg/kg i.p.), injected three times daily at 8-h intervals for 7 or 14 days, had a similar effect to that of bombesin on the SS mechanism of action. The combined administration of bombesin and its antagonist RC-3095 had a greater effect on the SS receptor-effector system than when administered separately. The present study indicates that the pancreatic SS receptor-effector system may be regulated by bombesin in vivo.