Identification of a novel AU-Rich element in the 3' untranslated region of epidermal growth factor receptor mRNA that is the target for regulated RNA-binding proteins.

The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated approximately 260-nucleotide (nt) cis-acting element in the 3' untranslated region (3'-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (~75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3' extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (~55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3' extended AU pentamer, but not the 5' extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3'-UTR that is bound by specific and EGF-regulated trans-acting factors. Furthermore, the 3' extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

Divergent ileal IGF-I and IGFBP-3 gene expression after small bowel resection: a novel mechanism to amplify IGF action?

Changes in the levels of ileal insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) mRNA in the rat following massive small bowel resection (MSBR) have been investigated with a sensitive S1 nuclease assay. IGF-I mRNA levels vary little over 7 days; in contrast IGFBP-3 mRNA levels decreased to one-third 7 h post-MSBR, and remained suppressed for the length of this study. We postulate that decreased ileal synthesis of IGFBP-3 enhances the ability of IGF-I to stimulate the adaptive response.

Expression of ileal glucagon and peptide tyrosine-tyrosine genes. Response to inhibition of polyamine synthesis in the presence of massive small-bowel resection.

Massive small-bowel resection results in a marked adaptive response in the residual terminal ileum. Increased polyamine synthesis is a necessary component of this response. The ileal L-cell-derived peptides enteroglucagon and peptide tyrosine tyrosine (PYY) have been implicated as humoral mediators of this response. We have previously reported a rapid and sustained increase in glucagon mRNA concentrations after massive small-bowel resection. In this study using an inhibitor of the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase, we have demonstrated that the response of the glucagon and PYY genes to massive small-bowel resection is dependent on polyamine biosynthesis. In addition, we have examined the response of both the ornithine decarboxylase and c-jun genes in this model of intestinal adaptation.

POU-domain gene expression in the gastrointestinal tract.

The process of self-renewal which occurs in the gastrointestinal epithelium is greatly amplified and accelerated during the intestinal adaptation which occurs in the residual ileum after massive small bowel resection (MSBR). As with growth and development, these processes must involve the coordinated regulation of many genes. Several families of nuclear proteins are known to be involved in the control of gene expression during development including the POU-domain genes; their expression has not been characterized in the gastrointestinal tract during normal cellular renewal or adaptation, and POU-domain encoding cDNAs were cloned from ileal RNA. Three known genes were cloned: Oct-1, Brn-1 and Tst-1 but no novel members of this gene family were identified. The encoded sequence for rat Oct-1 differs from that previously reported. Oct-1 is relatively ubiquitously expressed with increased expression during both development and adaptation. Minimal expression of Tst-1 was observed. Brn-1 exhibits limited expression in the adult gastrointestinal tract but may play a role in the fetal gastrointestinal tract.

Ileal proglucagon gene expression in the rat: characterization in intestinal adaptation using in situ hybridization.

BACKGROUND: Proglucagon-derived peptides are potential mediators of the adaptive response of the terminal ileum to massive small bowel resection. Ileal proglucagon messenger RNA (mRNA) levels increase during ileal adaptation. The present study explored the cellular basis of this response. METHODS: Sections of control ileum, ileum 4 days after resection, and pancreas were analyzed by in situ hybridization with 35S-labeled complementary RNA (cRNA) probes. RESULTS: Both the proglucagon and the peptide YY cRNA probes hybridized to discrete cells in the ileal mucosa, the disposition of which corresponds to that reported for intestinal L cells. Four days after resection there was a marked increase in the intensity of the signal for both probes without an increase in cell number. Insulin and histone H3 probes were used as controls to confirm the specificity of the hybridization seen with the L-cell specific, proglucagon, and peptide YY probes. CONCLUSIONS: The increase in proglucagon mRNA levels after massive small bowel resection is caused by an increase in the cellular content. The parallel increase in PYY mRNA levels implies an L cell--rather than a proglucagon gene--specific response.

Intracellular localization and metabolism of chylomicron remnants in the livers of low density lipoprotein receptor-deficient mice and apoE-deficient mice. Evidence for slow metabolism via an alternative apoE-dependent pathway.

The metabolism of chylomicron remnants in mice deficient in low density lipoprotein receptor (LDLr) or apolipoprotein E (apoE) was compared with that of control C57BL/6J mice. Mice were injected intravenously with chylomicron-like emulsions labeled with radioactive lipids. Blood samples were taken at fixed time intervals from the retro-orbital sinus, and clearance rates of the lipoproteins were assessed from the decline in plasma radioactivities. To follow the intracellular pathway of remnants in the liver, emulsions labeled with a fluorescent cholesteryl ester (BODIPY) were injected, and liver sections were processed and assayed by laser confocal microscopy. Catabolism of remnant cholesteryl esters was assessed by injecting emulsions labeled with cholesteryl[1-14C]oleate and measuring the expired CO2 from each animal. In apoE-deficient mice, remnant removal from plasma was totally impeded, while the clearance of remnants in LDLr-deficient mice was similar to that in C57BL/6J control mice. The confocal micrographs of livers 20 min after injection of fluorescent chylomicron-like emulsions showed evenly distributed fluorescent particles in the hepatocytes from control mice. In contrast, the fluorescent particles were mainly located in sinusoidal spaces in LDLr-deficient mice. Three hours after injection the livers from control mice showed few fluorescent particles, indicating that remnants have been catabolized, while the sections from LDLr-deficient mice were still highly fluorescent. Micrographs from apoE-deficient mice showed no fluorescent particles in the liver at any time after injection. Measurement of expired radioactive CO2 after injection of emulsions labeled in the fatty acid moiety of cholesteryl oleate indicated that remnant metabolism was slower in the LDLr-deficient mice and essentially nil in the apoE-deficient mice. Control mice had expired 50% of the injected label by 3 h after injection. We conclude that under normal circumstances, chylomicron remnants are rapidly internalized by LDLr and catabolized in hepatocytes, with a critical requirement for apoE. When LDLr is absent, remnants are taken up by a second apoE-dependent pathway, first to the sinusoidal space of the liver, with subsequent slow endocytosis and slow catabolism. Hepatic clearance via this second pathway is increased by heparin, inhibited by lactoferrin, heparinase, and suramin, and down-regulated by feeding a high fat diet.

The diurnal rhythms of cholesterol metabolism and plasma clearance of model chylomicrons: comparison of normal and genetically hypercholesterolemic rats (RICO).

Previous studies showed a slower clearance of cholesterol-labeled lymph chylomicrons in genetically hypercholesterolemic rats (RICO) compared with normocholesterolemic rats. In this study, we compared rates of lipolysis and remnant clearance in RICO versus control normocholesterolemic rats of the same strain (RAIF) or with control Wistar rats, by injecting chylomicron-like lipid emulsions labeled with 14C-triolein to trace lipolysis, and 3H-cholesteryl ester to trace remnant clearance. Our findings showed slower clearance of chylomicron remnants in RICO compared with control RAIF or with control Wistar rats. During the light period, the clearance of lipids from chylomicron-like lipid emulsions injected intravenously was significantly slower in RICO rats compared with normocholesterolemic control rats of the same strain, RAIF. Within the RICO group, clearance of emulsion triolein (TO) was faster during the dark period compared with the light period. In contrast, however, the clearance of the emulsion remnants traced by cholesteryl oleate (CO) was slower during the dark period. This behaviour was not found within the Wistar group, where the clearances of TO and CO were similar in the light and dark period. Hepatic clearance of chylomicron remnants is mediated primarily by the low density lipoprotein (LDL) receptor, the expression of which shows diurnal variation. In both Wistar and RICO rats, the expression of LDL receptors was highest during the dark period. The LDL receptors in hepatic microsomal membranes from RICO rats migrated faster on SDS polyacrylamide gel electrophoresis when compared with normal Wistar and the RAIF. However in hepatic plasma membranes the LDL receptors from RICO and Wistar rats appeared identical after immunoblotting. Furthermore the LDL receptors from RICO and Wistar rats responded similarly to treatment with neuraminidase. An alteration in post-translational processing of the LDL receptor could possibly account for the slower clearance of chylomicron remnants in the RICO.

cAMP controls human renin mRNA stability via specific RNA-binding proteins.

It is now recognized that post-transcriptional mechanisms are pivotal to renin production. These involve factors that modulate renin mRNA stability. In 2003 new data has emerged from work in Australia and Germany that has identified several of the, as many as, 20 or so proteins involved. These include CP1 (hnRNP E1), HuR, HADHB, dynamin, nucleolin, YP-1, hnRNP K and MINT-homologous protein. Cyclic AMP (cAMP) is a crucial regulator of renin secretion as well as transcriptional and post-transcriptional control of expression. Many of the RNA-binding proteins that were identified responded to forskolin, increasing in amount by two to 10-fold. The cAMP mechanisms that regulate renin mRNA target, at least in large part, other genes that presumably encode some of these proteins. The increase in the expression of these then facilitates, sequentially, renin mRNA stabilization and destabilization. Our data, using a battery of different techniques, confirm that CP1 and HuR stabilize renin mRNA, whereas HADHB causes destabilization. These proteins target cis-acting C-rich sequences (in the case of CP1) and AU-rich sequences (HuR) in the distal region of the 3'-untranslated region of renin mRNA. We found HADHB was enriched in juxtaglomerular cells and that that within Calu-6 cells HADHB, HuR and CP1 all localized in nuclear subregions, as well as cytoplasm (HADHB and CP1) and mitochondria (HADHB) commensurate with the role each plays in control of renin mRNA stability. The specific proteins that bind to human renin mRNA have begun to be revealed. Cyclic AMP upregulates the binding of several of these proteins, which in turn affect renin mRNA stability and thus overall expression of renin.

Features of cholesterol structure that regulate the clearance of chylomicron-like lipid emulsions.

Cholesterol is an absolute requirement for the clearance from plasma of the remnants of triglyceride-rich lipoproteins. Our laboratory previously established that cholesterol was essential for the hepatic uptake of remnant particles after intravenous injection of chylomicron-like lipid emulsions (1). The aim of the present study was to determine the structural features of the cholesterol molecule that regulate the metabolism of chylomicrons. Chylomicron-like lipid emulsions, which reflect the size and composition and mimic the physiology of lymph chylomicrons, were prepared with tracer amounts of labeled triolein ([14C]TO) and cholesteryl oleate ([3H]CO) to follow the hydrolysis of triglyceride and the uptake of chylomicron remnant particles by the liver. Sterols selected as cholesterol congeners with functional group variations were incorporated into the emulsions in place of cholesterol and injected intravenously in rats. Control emulsions contained either no cholesterol or approximately 1% (by weight) cholesterol. The effects of the different sterol structures on lipolysis and hepatic remnant uptake were compared with controls to determine the significance of various functional groups. Clearance of emulsion CO was impaired when cholesterol was absent or replaced by cholesteryl chloride, cholesteryl formate, or 3-keto-cholesterol. Clearance of emulsions containing epicholesterol, where the OH group at the 3-position is in the alpha configuration, was similar to control emulsions containing cholesterol. Congeners with an additional hydroxyl group, viz. 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or 25-hydroxycholesterol, reduced CO clearance. Androstenol, which lacks the side chain at the C17-position, also retarded CO clearance from plasma. In contrast, emulsions incorporating congeners with side chain variations such as campesterol, beta-sitosterol, stigmasterol, or saturated congeners of cholesterol such as cholestanol, coprostanol and its epimer, epicoprostanol, all were cleared similarly to emulsions containing cholesterol. In conclusion, for physiological clearance of a chylomicron-like emulsion, the presence of a hydroxyl (-OH) group at the 3-position and an alkyl side chain at the C17-position of cholesterol are essential, while the structure of the side chain and the saturation of the ring structure are not critical. The mechanism of the specificity of sterols on the metabolism of protein-free emulsions is unclear, but does not relate to changes in microfluidity of the surface lipids, nor to the amount or isoform of associated apolipoproteins.