Properties of the Complex Between Recombinant Human Progastrin and Ferric Ions

Binding of ferric ions to the hormone glycine-extended gastrin17 is essential for biological activity (Pannequin, J., et al. (2002). J. Biol. Chem. 277: 48602-48609). The aims of the current study were to determine the properties of the complex between recombinant human progastrin6-80 and ferric ions. The stoichiometry and affinity of ferric ion binding were determined by fluorescence spectroscopy. The selectivity of metal ion binding and the stability of the 59Fe(III) progastrin6-80 complex were determined by equilibrium dialysis. The stoichiometry of 2.5 +/- 0.1 moles Fe/mole progastrin, and the apparent dissociation constant of 2.2 +/- 0.1 microM, were similar to the values previously determined for glycine-extended gastrin17 at pH 4.0. Of the four trivalent and seven divalent metal ions tested, only ferrous and ferric ions bound to progastrin6-80. The ferric ion-progastrin complex was extremely stable, with a half-life of 117 +/- 8 days at pH 7.6 and 25 degrees C. We conclude that recombinant human progastrin6-80 selectively binds ferrous and ferric ions with high affinity in a stable 2:1 complex.     

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of ¹²⁵I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC₅₀ values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca²⁺]_i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca²⁺]_i but inhibited the [Ca²⁺]_i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.     

Naming progastrin-derived peptides

The antral hormone gastrin continues to be in focus, because its hormonal and growth promoting effects are essential both for the function of the normal stomach and for the pathogenesis of major dyspeptic and neoplastic diseases. Deduction of the progastrin structure has improved the insight in the cellular synthesis of gastrin, but has also revealed that the biosynthetic machinery is complex, and, accordingly, that progastrin is processed to a multitude of more or less bioactive fragments. The naming of these fragments has, however, become inconsistent and confusing. Therefore, we propose a systematic nomenclature for progastrin-derived peptides of which there are three classes: (I) The gastrins with the evolutionary preserved tetrapeptide amide (Trp-Met-Asp-PheNH₂) at the C-terminus, which ensures high-affinity binding to the gastrin (CCK-B) receptor. Among the gastrins, gastrin-34 and gastrin-17 constitute the primary forms. (II) Processing intermediates, which are early products of progastrin that contain the structure of the primary gastrins within their sequence, but still cannot bind the gastrin receptor due to insufficient processing at their C-terminus. (III) Flanking fragments from the N- and C-termini of progastrin that do not contain any primary gastrin in their sequence, but nevertheless may undergo posttranslational processing. Each fragment can be specified with suffixes corresponding to the derived sequence in progastrin.     

Divergent proliferative responses to a gastrin receptor ligand in synchronized and unsynchronized rat pancreatic AR42J tumour cells

Depending upon experimental model, the CCK-B/gastrin receptor ligand CI-988 exhibits either agonist or antagonist activity. To confirm that CI-988 behaves as an antagonist toward gastrin-stimulated growth, its effects on cell proliferation were investigated in unsynchronized and synchronized AR42J rat pancreatic tumour cells. In unsynchronized cultures CI-988 alone had no effect, but inhibited gastrin-stimulated cell proliferation. In contrast, in synchronized cultures, CI-988 stimulated cell proliferation. Similarly, CI-988 inhibited gastrin-stimulated cAMP production in unsynchronized cells, but stimulated cAMP formation in synchronized cultures. Therefore, CI-988 stimulation of cAMP production and proliferation in AR42J cell cultures appears to be cell cycle-dependent. CI-988 inhibited gastrin-stimulated intracellular calcium ([Ca2+]i) mobilization in both populations and thus acted as an antagonist toward this pathway. Because CCK receptor densities and affinities were similar in both cell populations, the data suggest that CI-988's divergent effects on cell proliferation are governed by postreceptor signalling events which vary with cell cycle.     

The Role of Gastrin and CCK Receptors in Pancreatic Cancer and other Malignancies

The gastrointestinal (GI) peptide gastrin is an important regulator of the release of gastric acid from the stomach parietal cells and it also plays an important role in growth of the gastrointestinal tract. It has become apparent that gastrin and its related peptide cholecystokinin (CCK) are also significantly involved with growth of GI cancers as well as other malignancies through activation of the cholecystokinin-B (CCK-B) receptor. Of interest, gastrin is expressed in the embryologic pancreas but not in the adult pancreas; however, gastrin becomes re-expressed in pancreatic cancer where it stimulates growth of this malignancy by an autocrine mechanism. Strategies to down-regulate gastrin or interfere with its interface with the CCK receptor with selective antibodies or receptor antagonists hold promise for the treatment of pancreatic cancer and other gastrin - responsive tumors.     

Altered influence of CCK-B/gastrin receptors on HDC expression in ECL cells after neoplastic transformation

Gastrin is one of the main factors controlling enterochromaffin-like (ECL) cell endocrine function and growth. Long-standing hypergastrinemia may give rise to ECL cell carcinoids in the gastric corpus in man and in experimental models. We have analysed the expression and function of CCK-B/gastrin receptors in normal ECL cells and in ECL cell tumours (gastric carcinoids) of the African rodent Mastomys natalensis. Hypergastrinemia induced by short-term (5 days) histamine2-receptor blockade (loxtidine) resulted in increased histidine decarboxylase (HDC) mRNA expression in the gastric oxyntic mucosa. This increase was significantly and dose-dependently reversed by selective CCK-B/gastrin receptor blockade (YM022). Long-term (12 months) hypergastrinemia, induced by histamine2-receptor blockade, gave rise to ECL cell carcinoids in the gastric oxyntic mucosa. CCK-B/gastrin receptor mRNA was only slightly elevated while HDC mRNA expression was eight-fold elevated in ECL cell carcinoids and was not influenced by CCK-B/gastrin receptor blockade. Thus CCK-B/gastrin receptor blockade of hypergastrinemic animals reduces the HDC mRNA expression in normal mucosa but not in ECL cell carcinoids. These results demonstrate that HDC mRNA expression in neoplastic ECL cells is not controlled by CCK-B/gastrin receptors.     

Rat progastrin processing yields peptides with altered potency at the CCK-B receptor

Details of prohormone processing patterns are revealed by purification and characterization of molecular forms stored in the tissues where the hormones are expressed. Molecular forms of rat gastrin were purified from antral extracts by gel permeation, anion exchange, and reverse-phase HPLC. Amidated and glycine-extended gastrins were detected with specific antisera and their structures determined by mass spectrometry. In rats, the only form shorter than gastrin-17 observed contained 16 amino acids. These data suggest that two enzymes process the amino terminus of gastrin-17. Pyrrolidone carboxylic acid peptidase removes the amino terminal pyrrolidone carboxylic acid (pyroGlu), forming gastrin-16. In mammals other than rat, gastrin-16 is then cleaved by dipeptidyl peptidase IV to form gastrin-14. In rat, this reaction does not take place because of proline residues Pro(2)-Pro(3)- in gastrin-16. Gastrin-16 is found in sulfated and nonsulfated forms and comprises 28% of the total gastrin immunoreactivity. Glycine-extended forms of gastrin-16 and gastrin-17 comprises 45% of the total gastrin immunoreactivity. The sulfated forms of gastrin-16 and gastrin-17 bind to the CCK-B receptor transfected into CHO cells with 10-fold higher affinity than the nonsulfated forms of these peptides. Therefore, processing of rat progastrin may modulate the expression of gastrin biological activity.     

PPARalpha agonists stimulate progastrin production in human colorectal carcinoma cells

The three subtypes of peroxisome proliferator activated-receptors (PPARalpha, delta and gamma) control the storage and metabolism of fatty acids. Treatment of rats with the PPARalpha ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARalpha ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARalpha ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARgamma agonist rosiglitazone had no significant effect on progastrin production. The PPARalpha ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARalpha ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.     

Growth of mouse hepatocytes is stimulated by gastrin

Hepatocyte growth is regulated by various growth factors, including epidermal growth factor (EGF) and insulin. Recently, several additional peptide hormones have been shown to stimulate growth of hepatocyte only in the presence of EGF or insulin and are thus termed secondary mitogens. Gastrin regulates growth of normal and neoplastic gastrointestinal tissues, but the effect on growth of hepatocyte is unknown. We examined the effect of gastrin on growth of a normal mouse hepatocyte (NMH) line established in our laboratory. Effect of gastrin-17 (G-17) (10^?8 to 10^?6 M) on growth of NMH cells was examined in either the presence or absence of EGF in the culture medium. Growth of NMH cells was evaluated by incorporation of either bromodeoxyuridine (BrdU) or ³H-thymidine and by counting cells. Presence of a cell-surface receptor for G-17 was determined by Scatchard analysis using ¹²⁵I-G-17. In the presence of EGF, gastrin stimulated growth of NMH cells; in the absence of EGF, gastrin did not affect growth. The stimulatory effect of gastrin on NMH cells was blocked by JMV 320, a CCK-B type receptor antagonist. NMH cells possess a single, high affinity binding site for gastrin (K_d = 1.2 nM); EGF increased the gastrin binding capacity compared to non-treated cells (3.5 ± 0.4 vs. 2.2 ± 0.6 fmol/10⁶ cells). G-17 stimulated growth of NMH cells through a single high affinity receptor for G-17 which pharmcologically appears to be the CCK-B type only in the presence of EGF and thus can be considered a secondary mitogen. © 1995 Wiley-Liss, Inc.     

Glycine-extended gastrin promotes the invasiveness of human colon cancer cells.

Colorectal cancers express significant amounts of immature glycine-extended gastrin (G-Gly) and G-Gly is able to stimulate cell proliferation in colonic cell lines and mucosa. Here we wished to investigate whether G17-Gly promote the invasiveness of LoVo human colonic cancer cells, a process which requires degradation of extracellular matrix by proteases and concomitant induction of cell migration. We confirmed that LoVo cells express gastrin and gastrin/CCK-B receptor mRNAs. We showed that these cells secrete matrix metalloproteinase (MMP)-1, -2, and -9. The function of MMP being to degrade components of extracellular matrix, they may thus favor cell migration. As compared to controls, G17-Gly (10(-7) to 10(-12) M) significantly enhanced about two to three times the LoVo cell migration through Matrigel, an artificial basement matrix barrier. Moreover, G17-Gly increased and gastrin/CCK-B receptor antagonists decreased MMP secretion in conditioned culture media of LoVo cells. Our findings show that physiological doses of incompletely processed form of gastrin induce the invasiveness of tumor cells in vitro and suggest a novel potential role for this peptide in the metastatic process of colonic cancers in vivo.     

Flow cytometric detection of progastrin interaction with gastrointestinal cells

The unprocessed gastrin precursor, progastrin (PG), is often overexpressed in colon cancer and other malignancies where it appears to stimulate colonic growth. Overexpression of progastrin also stimulates proliferation of normal colonic mucosa, but the receptors mediating these effects have not been identified. Here we report the development of a non-radioactive assay for assessment of PG binding to normal and transformed cells. Progastrin was labeled using biotinylation, and binding of biotinylated PG to cells was assessed using flow cytometry. Using this approach, we show strong and specific binding of PG to some cell lines (IEC-6, IEC-18, HT-29, COLO320) and minimal binding to others (HeLa, DC2.4, Jurkat). We also found PG binding to several non-gut epithelial lines, such as CHO-K1, COS-6 and HEK293 cells. The specificity of binding was confirmed by competition with cold, unlabeled PG but not with glycine-extended gastrin or amidated gastrin-17. Binding was not influenced by the presence of the classical CCK-2 receptor, but was partially dependent on the charged glycosaminoglycans (GAG). The analysis of primary colonic tissues isolated from wild type C57BL/6 mouse, revealed a small epithelial subpopulation of non-hematopoietic (CD45-negative) cells that strongly interacted with PG. Surprisingly, this population was greatly expanded in gastrin knockout mice. This non-radioactive, FACS-based assay should prove useful for further characterization of cells expressing the progastrin receptor.     

Mice overexpressing progastrin are predisposed for developing aberrant colonic crypt foci in response to AOM

Recent studies show that nonamidated gastrins (Gly-gastrin and progastrin) stimulate colonic proliferation. However, the role of nonamidated vs. amidated gastrins in colon carcinogenesis has not been defined. We measured intermediate markers of carcinogenesis in transgenic mice overexpressing either progastrin (hGAS) or amidated gastrin (INS-GAS) in response to azoxymethane (AOM). The hGAS mice showed significantly higher numbers of aberrant crypt foci (140-200% increase) compared with that in wild-type (WT) and INS-GAS mice (P < 0.05) after AOM treatment. The bromodeoxyuridine-labeling index of colonic crypts also was significantly elevated in hGAS mice vs. that in WT and INS-GAS mice. The results therefore provide evidence for a mitogenic and cocarcinogenic role of nonamidated gastrins (progastrin), which is apparently not shared by the amidated gastrins. Although nonamidated gastrins are now believed to mediate mitogenic effects via novel receptors, amidated gastrins mediate biological effects via different receptor subtypes, which may explain the difference in the cocarcinogenic potential of nonamidated vs. amidated gastrins. In conclusion, our results provide strong support for a cocarcinogenic role for nonamidated gastrins in colon carcinogenesis.     

Purification and structural characterization of progastrin-derived peptides from a human gastrinoma

Several peptides derived from the gastrin-predicted preprohormone sequence were isolated from a human gastrinoma by gel permeation, anion exchange, and reverse phase chromatography. The peptides were identified and characterized structurally by a combination of radioimmunoassays, mass spectral analysis, and microsequence analysis. The largest peptide, progastrin-(1-35) (cryptagastrin), extends from the putative processing site for the signal peptidase to the double basic residues adjacent to the amino terminus of gastrin 34. A shorter form of this peptide, progastrin-(6-35) (cryptagastrin-(6-35), was also isolated in smaller amounts. In addition, sulfated and nonsulfated gastrin 17 amides (progastrin-(55-71)) and the glycine-extended nonsulfated gastrin 17 (progastrin-(55-72)) were identified by radioimmunoassay, and their structures were confirmed by mass spectral analysis. Isolation of cryptagastrin indicates that the signal peptide of human preprogastrin contains 21 amino acid residues, and progastrin, therefore, contains 80 amino acids. There is minimal processing of the cryptic peptide preceding the sequence of gastrin 34. An amidated gastrin form larger than gastrin 34 could contain 71 amino acids. No evidence was obtained for processing that would produce gastrins containing more than 34 but less than 71 amino acid residues.     

Ferric ions are essential for the biological activity of the hormone glycine-extended gastrin

Amidated and nonamidated gastrins elicit different biological effects via distinct receptors in different tissues. Amidated gastrin 17 stimulates gastric acid secretion and the development of gastric carcinoids, whereas glycine-extended gastrin 17 stimulates proliferation of the colonic mucosa and the development of colorectal cancers. Because glycine-extended gastrin 17 binds two ferric ions with high affinity (Baldwin, G. S., Curtain, C. C., and Sawyer, W. H. (2001) Biochemistry 40, 10741-10746), we have investigated the identity of the iron ligands and the role of ferric ions in biological activity. Here we report the solution structure of glycine-extended gastrin 17, determined by NMR spectroscopy. The spectral changes observed upon the addition of ferric ions revealed that Glu(7) acted as a ligand at the first ferric binding site, and that Glu(8) and Glu(9) acted as ligands at the second ferric ion binding site. Fluorescence quenching experiments confirmed that a GglyE7A mutant bound only one ferric ion. The inability of this mutant to stimulate proliferation or migration in the IMGE-5 cell line and the observation that the iron chelator desferrioxamine selectively blocked the effects of glycine-extended gastrin 17 indicated that binding of a ferric ion to Glu(7) was essential for biological activity. This is the first report of an essential role for a metal ion in the action of a hormone.     

Interleukin-1beta up-regulates RGS4 through the canonical IKK2/IkappaBalpha/NF-kappaB pathway in rabbit colonic smooth muscle

Cellular synthesis of peptide hormones requires PCs (prohormone convertases) for the endoproteolysis of prohormones. Antral G-cells synthesize the most gastrin and express PC1/3, 2 and 5/6 in the rat and human. But the cleavage sites in progastrin for each PC have not been determined. Therefore, in the present study, we measured the concentrations of progastrin, processing intermediates and alpha-amidated gastrins in antral extracts from PC1/3-null mice and compared the results with those in mice lacking PC2 and wild-type controls. The expression of PCs was examined by immunocytochemistry and in situ hybridization of mouse G-cells. Finally, the in vitro effect of recombinant PC5/6 on progastrin and progastrin fragments containing the relevant dibasic cleavage sites was also examined. The results showed that mouse G-cells express PC1/3, 2 and 5/6. The concentration of progastrin in PC1/3-null mice was elevated 3-fold. Chromatography showed that cleavage of the Arg(36)Arg(37) and Arg(73)Arg(74) sites were grossly decreased. Accordingly, the concentrations of progastrin products were markedly reduced, alpha-amidated gastrins (-34 and -17) being 25% of normal. Lack of PC1/3 was without effect on the third dibasic site (Lys(53)Lys(54)), which is the only processing site for PC2. Recombinant PC5/6 did not cleave any of the dibasic processing sites in progastrin and fragments containing the relevant dibasic processing sites. The complementary cleavages of PC1/3 and 2, however, suffice to explain most of the normal endoproteolysis of progastrin. Moreover, the results show that PCs react differently to the same dibasic sequences, suggesting that additional structural factors modulate the substrate specificity.