Oncogenic ras induces gastrin gene expression through activation of the Raf-MEK-ERK signal transduction pathway.

Expression of gastrin, a gut hormone and growth factor, has tissue-specific transcriptional regulation and can be induced in some tumors. Previous studies have shown that a CACC cis-regulatory element is important for transcriptional activation in pancreatic insulinoma cells. To identify CACC-binding proteins, a phage cDNA library derived from a rat insulinoma cell line, RIN 38A, was screened by a Southwestern method. A novel member of the Cys 2 -His 2 zinc finger gene family was cloned and designated RIN ZF, having a cDNA sequence of 3.8 kilobases. One full-length and a shorter splice variant were sequenced and had predicted protein masses of 91.6 and 88.7 kDa. Expression of both splice forms were ubiquitous in fetal and adult rat tissues. Recombinant RIN ZF protein exhibited sequencespecific binding to the gastrin CACC element in a gel mobility shift assay. In transient transfections, both splice variants appeared to have only weak activating effects on gastrin-luciferase reporter gene transcription. Furthermore, RIN ZF coexpression with Sp1 appeared to block the strongly activating effects of Sp1 mediated through the CACC element. These findings suggest that a novel set of zinc finger proteins may help regulate gastrin gene expression by interfering with Sp1 transactivation.

When deprived of D-glucose for 24 hr, chicken embryo fibroblasts exhibit a marked increase in hexose transport activity compared with that of control cells. Scatchard analysis of [3H]cytochalasin B binding to starved cell plasma membranes (46 pmol/mg) indicated a six-fold increase compared with fed cell plasma membranes (7.5 pmol/mg). Irradiation of starved cell plasma membranes with high-intensity UV light in the presence of0.5 IAM [3H]cytochalasin B resulted in covalent labeling ofpolypeptides of Mr 52,000 and 46,000. In fed cell plasma membranes irradiated under the same conditions, both polypeptides were labeled but at greatly decreased levels. In fact, labeling of the Mr 52,000 polypeptide was barely detectable. The amount of D-glucose-sensitive [3H]cytochalasin B covalent insertion into these membrane components was increased 11 ± 2 (n = 4)-fold in starved versus fed cell plasma membranes. Photoaffinity labeling of both polypeptides in starved cell plasma membranes was inhibited by D-glucose, 3-O-methylglucose, 2-deoxyglucose, cytochalasin B, and cytochalasin A but not by D-sorbitol, L-glucose, or cytochalasin E. Half-maximal inhibition of labeling of the Mr 52,000 polypeptide occurred at 8 mM D-glucose whereas, for the Mr 46,000 polypeptide, half-maximal inhibition occurred at 40mM D-glucose. It is concluded that (i) two hexose transport proteins, one of Mr 46,000 and one of Mr 52,000, have been identified in chicken embryo fibroblasts and (ii) the increased affinity labeling of these transporter components after cell starvation may reflect increased numbers of transporters in the plasma membrane.The facilitated D-glucose transport systems found in adipose tissue, skeletal and heart muscle, and various cultured cells can be regulated in response to hormones and cellular nutritional states. Hamster and chicken embryo fibroblasts maintained in D-glucose-deprived culture medium for 24 hr have greatly elevated levels of hexose transport activity compared with that of normally fed cells (1-6). This increase in transport activity due to D-glucose starvation can be prevented by protein synthesis inhibitors (1-4, 7-10). Addition of D-glucose to the starved cell culture medium results in a gradual decrease in the elevated transport activity back to the level found in normally maintained D-glucose-fed cultures (4, 10). When cycloheximide and D-glucose are added simultaneously to the starved cell culture medium, the elevated transport activity decreases at a much slower rate than observed in the presence of D-glucose alone (10). These observations suggest that the nutritional state of the cell regulates the relative rates of synthesis and degradation of the transport system component(s).Further clarification of transport regulatory mechanisms requires a method to specifically label or quantitate (or both) the hexose transporters. One such method is to use cytochalasin B, which is a potent reversible inhibitor of hexose transport in a number of animal cells (11) and appears to be a competitive antagonist (12...

The gastrin gene is transiently expressed in fetal pancreatic islets during islet neogenesis but then switched off after birth when islet cells become fully differentiated. Previous studies identified a cis-regulatory sequence between -109 and -75 in the human gastrin promoter which binds islet cell-specific activators and a nonspecific repressor and thus may act as a molecular switch. The present study identified another cis-regulatory sequence (-163ACACTAAATGAAAGGGCGGGGCAG-140) which bound two islet nuclear proteins in a mutually exclusive manner, as defined by gel shift competition, methylation interference, and DNase I foot-printing assays. The general transactivator Sp1 recognized the downstream GGGCGGGG sequence, but Sp1 binding was prevented when another islet factor bound to the adjacent AT-rich sequence (CTAAATGA). This gastrin AT-rich element is nearly identical to the binding site (ATAAATGA) for the islet-specific transcription factor beta TF-1. However, the gastrin AT-binding factor appeared to differ from beta TF-1 in its gel mobility shift pattern. Transfections of rat insulinoma cells revealed that mutations which blocked binding to the AT-rich element but allowed Sp1 binding up-regulated transcriptional activity. These results suggest that the gastrin AT-binding factor blocks transactivation by Sp1 and may have a role in the repression of gastrin transcription seen at the end of islet differentiation.