Biomedical subjects

J L Merchant

Publications and source records attributed to J L Merchant.

34 records · Page 2Linked to original sources

PubMed1997

ZBP-89, a Krüppel-type zinc finger protein, inhibits cell proliferation.

ZBP-89 is a Krüppel-type zinc finger transcription factor that binds to GC-rich sequences. Overexpression of this factor prevents EGF induction of the gastrin promoter; therefore, we postulated that ZBP-89 may modulate cellular proliferation. To test this hypothesis, ZBP-89 was overexpressed in immortalized (GH4) and malignant (AGS) cell lines. Growth parameters, e.g., 3H-thymidine, BrdU labeling, flow cytometry and ornithine decarboxylase promoter activity were analyzed. The results show that DNA synthesis is inhibited and progression to S phase is blocked in GH4 cells. Collectively, these studies demonstrate that ZBP-89 inhibits cellular proliferation at least in part through its ability to bind and repress ornithine deacarboxlyase promoter activity.

Adenocarcinoma↗

PubMed1997

RAP1-like binding activity in islet cells corresponds to members of the Sp1 family of transcription factors.

Deletion and mutational analyses of the gastrin promoter have identified a binding site for the yeast transcription factor RAP1 relevant for transcriptional activation in islet cells. We here report that the mammalian transcription factors binding to this site in islet cells are the Sp transcription factor members Sp1 and Sp3. Furthermore, functional analyses revealed Sp1- and Sp3-mediated transcriptional activation of gastrin. These data reveal that the zinc finger proteins Sp1 and Sp3 do have similar binding specificities as the multifunctional yeast RAP1 protein.

Animals↗

PubMed1997

Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis.

Sp1 nuclear levels have been shown to directly correlate with the proliferative state of the cell. We therefore studied changes in the abundance of Sp1 in a rat pituitary cell line GH4 whose growth rate is regulated by epidermal growth factor (EGF). Nuclear extracts from GH4 cells treated with 10 nM EGF for at least 16 h showed a 50% decrease in Sp1 binding to a GC-rich element present in the gastrin promoter. The decrease in binding correlated with a decrease in cell proliferation, a loss of nuclear Sp1 protein and a 50-60% decrease in Sp1-mediated transactivation through an Sp1 enhancer element in transfection assays. Okadaic acid, a phosphatase inhibitor, was synergistic with the effect of EGF on Sp1 protein levels suggesting that the loss of Sp1 was mediated by phosphorylation events. This result was confirmed by showing a 2-fold increase in orthophosphate-labeled Sp1 with EGF and okadaic acid. Cycloheximide prevented the expected loss of Sp1 mediated by EGF and okadaic acid suggesting that the synthesis of a protease may mediate these events. This hypothesis was tested directly by showing that the cysteine protease inhibitor leupeptin prevented Sp1 degradation. Using the PEST-FIND computer program, the computed PEST score for human and rat Sp1 is 10.4 and 13.7, respectively, indicating that Sp1 has a domain with a high concentration of proline, glutamic acid, serine, and threonine residues as reported for a number of proteins with inducible rates of degradation. Collectively, these results indicate that sustained stimulation of GH4 cells by EGF initiates a cascade of phosphorylation events that promotes Sp1 proteolysis, decreased Sp1 nuclear levels and decreased cellular proliferation.

Amino Acid Sequence↗

PubMed1997

Transient transcriptional activation of gastrin during sodium butyrate-induced differentiation of islet cells.

Transient expression of pancreatic gastrin corresponds to a period of rapid islet cell development. After birth gastrin expression silencing is coincidental with islet cell terminal differentiation, while persistent expression is accompanied with nesidioblastosis and reexpression observed in islet cell tumors. Experiments with transgenic animals suggested that gastrin might act synergistically with growth factors to stimulate islet cell development. The present study intended to establish an in vitro cell culture model to analyse the molecular events controlling gastrin gene activation and repression dependent on islet cell differentiation. Sodium butyrate, a proliferation-arresting compound has previously been shown to differentiate insulinoma cells while increasing insulin production. The present paper demonstrates concomitant transient increase in gastrin mRNA, intracellular and secreted gastrin during sodium butyrate treatment. Increased gastrin expression was due to activation or derepression of gastrin promoter activity as revealed by promoter analyses. This in vitro model mimics the expression pattern of gastrin and insulin observed during fetal islet cell development and provides an excellent tool to analyse the molecular mechanisms controlling gastrin gene activation and selective repression during islet cell differentiation.

Animals↗

PubMed1997

EGF receptor activation stimulates endogenous gastrin gene expression in canine G cells and human gastric cell cultures.

Gastrin release from the antral gastrin-expressing cell (G cell) is regulated by bombesin and luminal factors. Yet, these same extracellular regulators do not stimulate expression of the gene. Since the gastric mucosa expresses large quantities of EGF receptor ligands such as TGFalpha, we examined whether EGF receptor ligands stimulate gastrin gene expression in gastrin-expressing cell cultures. EGF receptor activation of primary cultures stimulated gastrin gene expression about twofold; whereas bombesin treatment of antral G cell cultures stimulated gastrin release but not gene expression. EGF and TGFalpha were weak stimulants of gastrin release. EGF receptor activation of AGS human gastric adenocarcinoma cell line stimulated gastrin gene expression nearly fourfold; and gastrin reporter constructs transfected into AGS cells were stimulated more than fourfold by EGF. EGF induction was conferred by the previously defined GC-rich gastrin EGF response element (gERE) element located at -68 to -53 bp upstream from the cap site since a mutation of the gERE element abolished both basal and EGF induction. Moreover, EGF treatment of AGS cells stimulated binding of the transcription factor Sp1 to this element. Collectively, these results demonstrate that gastrin gene expression and gastrin release are regulated by different signaling pathways: gene expression by EGF receptor activation and gastrin secretion by neuropeptides and luminal factors.

Animals↗

PubMed1997

Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.

ZBP-89 is a Krüppel-type zinc finger protein that binds to the gastrin EGF response element (gERE). Sp1 binds to the same DNA element and transactivates gastrin promoter activity, whereas ZBP-89 competes for Sp1 binding and prevents EGF induction. Both transcription factors mediate growth factor signals originating from the EGF receptor and thus were studied in normal and neoplastic tissues or cell lines. When compared to normal tissue from the same patient, ZBP-89 protein expression was increased in neoplastic tissue from the stomach antrum and in malignant cell lines. RT-PCR analysis of ZBP-89 mRNA correlated with protein overexpression. Immunocytochemical studies confirmed that ZBP-89 expression is elevated in neoplastic tissue and chronic gastritis, whereas Sp1 expression was nearly unchanged. These results suggest that the transcription factor ZBP-89, like Sp1, may be a marker for neoplastic transformation in some gastric cancers.

Cells, Cultured↗

PubMed1997

Gastrin regulates the human histidine decarboxylase promoter through an AP-1-dependent mechanism.

The histidine decarboxylase (HDC) gene is regulated transcriptionally by gastrin and phorbol 12-myristate 13-acetate (PMA) through a protein kinase C (PKC)-related pathway. To determine the role of AP-1 (fos/jun) in the regulation of the HDC promoter, gastric cancer (AGS-B) cells stably expressing the cholecystokinin-B/ gastrin receptor and the 1.8-kb human (h) HDC-luciferase (luc) construct were cotransfected with constructs expressing c-fos and c-jun. Overexpression of c-fos and c-jun activated the HDC promoter in a dose-dependent fashion in 1.8-kb hHDC-luc/AGS-B cells as well as in transfected F9 embryonal carcinoma cells, which lack endogenous AP-1 activity. PMA was unable to activate the HDC promoter in F9 cells, which were not transfected with c-fos and c-jun. Gastrin stimulation increased c-fos and c-jun mRNA abundance and AP-1-dependent transcriptional activity, as assessed by a reporter construct in which the CAT reporter gene is under the control of a 12-O-tetradecanoylphorbol-13-acetate response element multimer. Gastrin-stimulated HDC promoter activity was blocked by transfection of c-fos antisense and dominant negative c-jun expression constructs. Finally, overexpression of c-fos and c-jun activated the hHDC promoter through a downstream cis-acting element (gastrin response element), which does not bind AP-1. In conclusion, activation of AP-1 is essential for gastrin-stimulated HDC transcription, but the mechanism appears to be indirect.

Antisense Elements (Genetics)↗

PubMed1996

ZBP-89, a Krüppel-like zinc finger protein, inhibits epidermal growth factor induction of the gastrin promoter.

We have shown previously that a GC-rich element (GGGGCGGGGTGGGGGG) conferring epidermal growth factor (EGF) responsiveness to the human gastrin promoter binds Sp1 and additional undefined complexes. A rat GH4 cell line expression library was screened by using a multimer of the gastrin EGF response element, and three overlapping cDNA clones were identified. The full-length rat cDNA encoded an 89-kDa zinc finger protein (ZBP-89) that was 89% identical to a 49-kDa human factor, ht(beta), that binds a GTGGG/CACCC element in T-cell receptor promoters. The conservation of amino acids between the zinc fingers indicates that ZBP-89 is a member of the C2H2 zinc finger family subclass typified by the Drosophila Krüppel protein. ZBP-89 is ubiquitously expressed in normal adult tissues. It binds specifically to the gastrin EGF response element and inhibits EGF induction of the gastrin promoter. Collectively, these results demonstrate that ZBP-89 functions as a repressor of basal and inducible expression of the gastrin gene.

Amino Acid Sequence↗

PubMed1996

Fos is required for EGF stimulation of the gastrin promoter.

Gastrin gene expression is regulated by developmental cues, pH, and inflammation. These processes are mediated by various extracellular ligands, e.g., growth factors, cytokines, and neuropeptides that also stimulate c-fos gene expression. Therefore, to determine whether Fos is required for stimulation of the gastrin promoter, a c-fos sense expression vector was coexpressed with a gastrin reporter construct in a GH4 rat pituitary cell line. We found that epidermal growth factor (EGF) and tumor necrosis factor-alpha (TNF-alpha) transiently stimulate an increase in Fos protein that precedes stimulation of the gastrin promoter. However, the induction mediated by TNF-alpha was weaker than that mediated by EGF, indicating minimal overlap of the signaling pathways activated by EGF and TNF-alpha. Accordingly, overexpression of c-fos mRNA facilitated primarily EGF rather than TNF-alpha induction of the gastrin promoter. Expression of the c-fos gene in the absence of ligand did not stimulate the gastrin promoter. Thus c-fos gene expression is required but is not sufficient for induction of the gastrin promoter by EGF.

Animals↗

PubMed1995

Sp1 affinity for GC-rich elements correlates with ornithine decarboxylase promoter activity.

The highly conserved ornithine decarboxylase (E.C.4.1.1.17) promoter contains multiple binding sites for Sp1 within the first 400 bp of the cap site. Therefore the ability of individual Sp1 elements to confer transactivation alone or in combination was tested. We show that different Sp1 sites vary in their affinity for Sp1 and that increasing affinity correlates with enhancer activity. In addition, several adjacent Sp1 sites synergistically enhanced promoter activity. Thus, the strength of promoter transactivation correlated with both the number of GC-rich elements and their affinity for Sp1 protein.

Adenoma↗

PubMed1995

Epidermal growth factor stimulation of the human gastrin promoter requires Sp1.

Growth factors coordinately regulate a variety of different genes to stimulate cellular proliferation. In the stomach, gastrin, epidermal growth factor (EGF), and transforming growth factor-alpha all mediate gastric mucosal homeostasis by promoting cell renewal. We have previously shown that EGF and phorbol esters stimulate the human gastrin promoter through a novel GC-rich DNA element 5'-(68)GGGGCGGGGTGGGGGG-53 called gERE (gastrin EGF response element). In this report, we show that three factors bind to this element, the transcription factor Sp1 and two fast migrating complexes designated gastrin EGF response proteins (gERP 1 and 2). To understand how these factors bind and confer EGF responsiveness, mutations of gERE were tested in vitro for protein binding and in vivo for promoter activation. Both gel shift assays and UV cross-linking studies revealed that the factors bind to overlapping domains, Sp1 to the 5' half-site and gERP 1 and 2 to the 3' half-site. Placing either the 5' or 3' mutations upstream of a minimal gastrin promoter abolished EGF induction. Therefore both the 5' and 3' domains were required to confer EGF induction. Collectively, these results demonstrate that complex interactions between Sp1 and other factors binding to overlapping gERE half-sites confer EGF responsiveness to the gastrin promoter.

Animals↗

PubMed1995

cAMP regulates gastrin gene expression.

Gastrin is one of the most potent regulators of acid secretion and gastrointestinal cell growth. A variety of signals regulate gastrin release from the antral G cell. However, whether these secretagogues also stimulate gastrin gene expression has not been established. Dramatic increases in gastrin gene expression occur in the stomach after birth and in response to chronic achlorhydria. Moreover, gastrin gene expression in malignant islet cell tumors (gastrinomas) appears to represent reactivation of the fetal pattern of expression in the pancreas. Thus differential expression of the gastrin gene is a reflection of differences in transcriptional control. Since various luminal and humoral factors stimulate the production of cAMP and gastrin secretion in both gastrinoma and antral G cells, we studied the effect of cAMP on gastrin gene expression. Using stable and transient transformants of a pituitary cell line containing the human gastrin gene, we found that cAMP stimulates a three-fold increase in gastrin mRNA levels and that the response maps to elements located between -148 and -40 base pairs upstream from the cap site. Collectively, these studies link an important regulator of gastrin secretion to regulation of gastrin gene expression.

Base Sequence↗

PubMed1991

A GC-rich element confers epidermal growth factor responsiveness to transcription from the gastrin promoter.

Epidermal growth factor (EGF) and transforming growth factor alpha are important determinants of mucosal integrity in the gastrointestinal tract, and they act both directly and indirectly to prevent ulceration in the stomach. Consistent with this physiological role, EGF stimulates transcription of gastrin, a peptide hormone which regulates gastric acid secretion and mucosal growth. EGF stimulation of gastrin transcription is mediated by a GC-rich gastrin EGF response element (gERE) (GGGGCGGGGTGGGGGG) which lies between -54 and -68 in the human gastrin promoter. The gERE sequence also confers weaker responsiveness to phorbol ester stimulation. The gERE sequence differs from previously described EGF response elements. The gERE DNA sequence specifically interacts with a GH4 DNA-binding protein distinct from previously described transcription factors (Egr-1 and AP2) which bind GC-rich sequences and mediate transcriptional activation by growth factors. Furthermore, the gERE element does not bind the Sp1 transcription factor even though the gERE sequence contains a high-affinity Sp1-binding site (GGCGGG).

Animals↗

PubMed1987

Characterization and use of polyclonal antibody to Na+,K+-ATPase: immunocytochemical localization in salt glands of the duck.

The amount of Na+,K+-ATPase of the avian salt gland increased concomitantly with plasma membrane surface area during salt feeding of ducklings (adaptation), and both enzyme content and membrane surface area decreased upon return to fresh water (deadaptation). In a further study of the enzyme, a marker for plasma membrane biogenesis, polyvalent antibodies were raised to the denatured alpha-subunit of the purified ATPase. Antisera did not inhibit enzymatic activity but immunoprecipitated the phosphorylated intermediate of the alpha-subunit. Furthermore, the alpha-subunit, which was not glycosylated, was immunoprecipitated from homogenates of tissue slices metabolically labelled with [35S]-methionine, using antisera raised against either duck salt gland or dog kidney alpha-subunit. The former antisera also recognized the alpha-subunit in the brain, heart, kidney, liver, intestine and skeletal muscle of the duck. Immunocytochemistry with the antisera raised to the duck salt gland alpha-subunit revealed reaction at basolateral as well as apical plasma membrane in the duck salt gland principal cells, with essentially no deposits on peripheral cells, fibroblasts, erythrocytes, endothelial cells and neural elements. Within the principal cells, immunolabelling was also detected on small vesicles, multivesicular bodies and lysosomes; deposits on extracellular debris and vesicles in the lateral and lumenal spaces were also apparent. The labelling patterns were qualitatively but not quantitatively similar in salt glands of control, adapted and deadapted ducklings, and are discussed in the context of a model for plasma membrane biogenesis and turnover in which degradative events may play a major role.

Adaptation, Physiological↗

PubMed1985

Correlation of Na+,K+-ATPase content and plasma membrane surface area in adapted and de-adapted salt glands of ducklings.

During salt-water adaptation, an increase occurs in Na+,K+-ATPase content and surface area of the basolateral plasma membrane of the principal cell of the duck salt gland. To determine the degree to which these changes are correlated, accepted morphometric methods were used to determine numerical cell densities and plasma membrane surface densities of peripheral and principal cells. After adaptation, the plasma membrane surface area per principal cell was five times greater than in controls. Following de-adaptation, the plasma membrane content in principal cells returned to 1.9 times control levels. Two other cell constituents, mitochondria and lipid droplets, displayed similar quantitative changes. Na+,K+-ATPase content increased about fourfold with adaptation and decreased to near control levels with de-adaptation. Thus, changes in Na+,K+-ATPase content and basolateral plasma membrane surface area in adapting and de-adapting secretory epithelia of the salt gland occur nearly in parallel. These quantitative data enable Na+,K+-ATPase synthesis and degradation to be investigated in relation to membrane biogenesis.

4-Nitrophenylphosphatase↗

PubMed1977

3-Hydroxy-3-methylglutaryl coenzyme A reductase in isolated villous and crypt cells of the rat ileum.

The localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34) in the villous and crypt cells of the small intestine was accomplished after separating these cells from the mucosal layer by sequential dissociation in a "dual-buffer" system. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villous cell, and thymidine kinase, specific to the crypt cell. Cells obtained were 95-100% viable, and no relative difference in lability was observed, as evidenced by the equal distribution of acid phosphatase. This method of cell separation was an improvement over the "scraping" technique which damaged cells severely and produced villous preparations that contained little or no reductase activity. The HMG-CoA reductase specific activity in whole cell homogenates of the ileal villi was 0.47 and of the crypts was 0.27 nmol/min per mg of protein, considerably higher values than have been reported earlier. Also in comparison to the crypts, the villi incorporated 1.5-fold more [(14)C]-acetate into sterols, a ratio similar to that describing the distribution of HMG-CoA reductase in the two cell populations. These results unequivocally establish that the villi have higher HMG-CoA reductase activity than the crypts and confirm an earlier report from this laboratory that the villi are a major site of sterol synthesis. The sterol bio-synthetic capacity of the small intestine was highest in the ileum and decreased towards the jejunum. The HMG-CoA reductase specific activity of the ileum averaged 0.30 and that of the jejunum 0.10 nmol/min per mg of protein; however, the cholesterol content of the ileum was slightly lower than the jejunum. These results are discussed to suggest the possibility that the sterol content of the ileum may largely be due to in situ synthesis.

Acetates↗