Cyclin D1 expression is regulated by the retinoblastoma protein.

The product of the retinoblastoma susceptibility gene, pRb, acts as a tumor suppressor and loss of its function is involved in the development of various types of cancer. DNA tumor viruses are supposed to disturb the normal regulation of the cell cycle by inactivating pRb. However, a direct function of pRb in regulation of the cell cycle has hitherto not been shown. We demonstrate here that the cell cycle-dependent expression of one of the G1-phase cyclins, cyclin D1, is dependent on the presence of a functional Rb protein. Rb-deficient tumor cell lines as well as cells expressing viral oncoproteins (large tumor antigen of simian virus 40, early region 1A of adenovirus, early region 7 of papillomavirus) have low or barely detectable levels of cyclin D1. Expression of cyclin D1, but not of cyclins A and E, is induced by transfection of the Rb gene into Rb-deficient tumor cells. Cotransfection of a reporter gene under the control of the D1 promoter, together with the Rb gene, into Rb-deficient cell lines demonstrates stimulation of the D1 promoter by Rb, which parallels the stimulation of endogenous cyclin D1 gene expression. Our finding that pRb stimulates expression of a key component of cell cycle control, cyclin D1, suggests the existence of a regulatory loop between pRb and cyclin D1 and extends existing models of tumor suppressor function.     

Vascular endothelial growth factor expression in human endometrium is regulated by hypoxia

Endometrial growth and repair after menstruation are associated with profound angiogenesis. Abnormalities in these processes result in excessive or unpredictable bleeding patterns and are common in many women. It is therefore important to understand which factors regulate normal endometrial angiogenesis. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen that plays an important role in normal and pathological angiogenesis. In this study we show that expression of VEGF is regulated by hypoxia in human endometrium. Culture in vitro for 24 h under hypoxic conditions resulted in a 2- to 6-fold increase in VEGF secretion by both stromal and epithelial cells isolated from human endometrium. Quantitative RT-PCR was used to measure VEGF messenger ribonucleic acid (mRNA) levels in these cells. After hypoxia, VEGF mRNA levels increased 1.8-fold in stromal cells and 3.4-fold in glandular epithelial cells. The mRNA for each VEGF splice variant increased to an equal extent. The increase in VEGF secretion by stromal and epithelial cells in response to hypoxia was not altered by treatment at the same time with estradiol or progesterone. In situ hybridization of human endometrium during menstruation, when steroid levels are low but the tissue is subject to ischemia, showed strong hybridization to VEGF mRNA in both stromal and glandular cells. These results show that local factors, such as hypoxia, can regulate VEGF expression in the endometrium. This may play an important part in normal endometrial repair after menstruation. The secretion of VEGF by endometrial cells under hypoxic conditions may also be important in the pathogenesis of endometriosis, because it would be predicted to assist revascularization of desquamated endometrial explants when they attach at ectopic sites.     

On the mediation inflammatory reaction in the human gallbladder epithelium.

Human gallbladder epithelium was homogenized with a view to maintaining the integrity of subcellular components. In such homogenates, N-acetyl-beta-glucosaminidase, beta-glucuronidase, beta-galactosidase, beta-glucosidase, beta-fucosidase, beta-xylosidase, and acid phosphatase were demonstrated together with phospholipase activity. All the enzymes exhibited structure-linked latency. After discarding cellular debris from the homogenate, remaining subcellular organelles were analytically separated by density gradient centrifugation. After 100,000 g for 1 hour, particles containing acid glycosidases were recovered at a sucrose density of 1.18-1.19, whereas the mitochondrial marker enzyme succinate-reductase accumulated at a density of 1.16. The bulk of sedimentable phospholipase activity was recovered with particles sedimenting at 1.18-1.19. The results are interpreted as indicating that phosphalipase is present in lysosomes of the human gallbladder epithelium. Release of acid hydrolases, in lysosomes of the human gallbladder epithelium. Release of acid hydrolases, in lysosomes of the human gallbladder epithelium. Release of acid hydrolases, particularly phospholipase A, from the gallbladder epithelium is discussed as mediation of an inflammatory reaction in the gallbladder, i.e. cholecystitis.     

Differences in antigen expression between neoplastic and nonneoplastic gallbladder epithelium

Immunoreactivity for a panel of 15 monoclonal antibodies (MAbs), which are known to react with different gastrointestinal tumor antigens, was assessed in formalin-fixed paraffinembedded sections that were prepared from cholecystectomy specimens obtained from Mexican patients. Each case was classified histologically into one of the following groups: (1) invasive adenocarcinoma (N=21), (2) high-grade dysplasia (carcinomain situ) (N=2), (3) low-grade dysplasia (N=4), hyperplasia (4) (N=15), and (5) chronic cholecystitis (N=10). Significant differences (P<0.05) were identified among the five histopathologic groups in the proportion of epithelial cells demonstrating immunoreactivity with MAbs to Lewis^b; Lewis^a; sialylated Lewis^a; sialylated Lewis^a and Lewis^a; Y antigen; H antigen; X antigen; X-like antigen; 200-kDa protein of CEA; 180-, 160-, 50-, 40-kDa proteins of CEA; 30- to 37-kDa protein; and an undefined antigen identified by MAb 99–57, with invasive carcinoma more frequently being positive as compared to nonneoplastic (hyperplasia, chronic cholecystitis) epithelium. Significant differences were also observed among the five histopathologic groups (P<-0.0005) in the proportion of epithelial cells demonstrating immunoreactivity with MAbs to Y antigen and the 20- to 50-kDa glycoprotein. However, with these two antibodies immunoreactivity was more frequently found in nonneoplastic epithelium rather than in invasive carcinomas. No significant differences in immunoreactivity were detected among the different histologic groups with MAb to blood group B antigen, types 1 and 2. This study demonstrates that cellular antigens are both developed and lost during the process of neoplastic transformation in the gallbladder. Future studies including a greater number of cases with varying degrees of gallbladder intraepithelial neoplasia are necessary in order to be able to draw meaningful conclusions regarding the antigenic expression of epithelium at different stages of tumorigenesis.Presented at the Annual Meeting of the American Gastroenterological Association, San Francisco, California, 1986.This study was supported by grants CA 35934 and CA 25874 from the National Cancer Institute.     

Placental glucose transporter expression is regulated by glucocorticoids

Although glucocorticoids play important roles in development and fetal programming, they are widely used for treatment of a variety of diseases during pregnancy. In various tissues, glucocorticoids down-regulate glucose transport systems; however, their effects on glucose transporters in the placenta are unknown. In the present study, the glucose carrier proteins GLUT1 and GLUT3 were localized in the trophoblast and endothelium of the human, rat, and mouse placenta. Subsequently, it was investigated whether glucocorticoids affect messenger ribonucleic acid and protein expression of these molecules by Northern and Western blotting using 1) human term placental trophoblast cells cultured in the presence or absence of 0.5, 5, and 50 micromol/L triamcinolone; 2) placentas of rats that received a single i.p. dose of 0.38 mg/kg triamcinolone; and 3) placentas of transgenic mice bearing an antisense glucocorticoid receptor gene construct. In all of these systems, both glucose transporters were significantly down-regulated (P < 0.05), with the exception of increased GLUT3 messenger ribonucleic acid and protein levels in transgenic mice. The results demonstrate that triamcinolone is a potent regulator of placental GLUT1 and GLUT3 expression involving the glucocorticoid receptor. We speculate that impaired expression of placental glucose transporters after glucocorticoid administration might contribute to the adverse side-effects, the foremost of which is a growth-retarded fetus, of this treatment during pregnancy.     

Cell proliferation in the human gallbladder epithelium: effect of distension.

DNA synthesis activity in the epithelium of the human gallbladder was studied through in vitro labelling of mucosal specimens with 3H-thymidine and autoradiography. The specimens were taken at the time of a surgical operation. Eight 'normal' gallbladders and six distended gallbladders from patients with carcinomatous obstruction of the common bile duct were examined. Proliferative activity was very low in the normal and significantly higher in the distended gallbladders.     

Mucin gene expression in gallbladder epithelium with black pigment stone ascertained by in situ hybridization

Black pigment stones are usually found in patients with liver cirrhosis or hemolytic disease. Mucoglycoproteins are present in a significant amount in black pigment stones and contribute to the matrix of gallstones. Epithelium of stone-containing gallbladders contains much more mucin than those without stones. In this study, we try to determine by in situ hybridization the mucin gene expression in black stone-containing gallbladders and try to find the diversity of mucin gene expression in gallbladders containing black pigment stones and those without stones. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of gallbladders with black pigment stones (n = 10) and those without stones (n = 6) to identify the expression of MUC1, MUC2, MUC3, MUC4, MUC5B and MUC6 in gallbladder epithelium. The findings showed that (1) mRNA expression of MUC1, MUC3, MUC5B and MUC6 were found in all gallbladders with black pigment stones, while they were expressed in 33.3%, 83.3%, 83.3% and 66.7% respectively in those without stones. They were expressed more strongly and extensively in gallbladders with stones when compared to those without stones. (2) MUC2 and MUC4 labeling were absent in gallbladders without stones, while they were present in 20% and 60% of gallbladders with black pigment stones, respectively. We conclude that MUC3, MUC5B and MUC6 were the main mucin gene expression in either gallbladder with or without stones. Altered mucin gene expression occurred in gallbladders with black pigment stones, such as the presence of MUC2 and MUC4 and increased expression of MUC1, MUC3, MUC5B and MUC6 in black stone-containing gallbladders. The higher incidence and stronger labeling intensity of mucin gene expression of MUC2, MUC3, MUC5B and MUC6 in black stone-containing gallbladder may reflect abundant mucin content in these gallbladders. Increased expression of MUC2 and MUC4 in black stone-containing gallbladder epithelium indicated that intestinal metaplasia and altered mucin genes could occur in diseased gallbladders.     

Adiponectin receptor gene expression in human skeletal muscle cells is not regulated by fibrates and thiazolidinediones

BACKGROUND: Thiazolidinediones as PPARgamma agonists and fibrates as PPARalpha agonists improve insulin sensitivity in insulin-responsive tissues. Recent data show an induction of adiponectin receptor 2 (AdipoR2) by PPARalpha and PPARgamma agonists in human macrophages. OBJECTIVE: In this study, we examined the effects of thiazolidinediones and fibrates on the expression of adiponectin receptors in human skeletal muscle cells, an important cell type in the context of insulin resistance. RESULTS AND METHODS: In vitro differentiated human myotubes treated with troglitazone or rosiglitazone (20 h) showed no significant changes in AdipoR1 and AdipoR2 mRNA expression. PPARgamma activation was controlled by determination of PPARgamma mRNA induction. Likewise, differentiated myotubes treated with Wy-14,643 or fenofibrate (20 h) revealed no significant regulation of AdipoR1 and AdipoR2 mRNA. PPARalpha activation was assessed by measuring PDHK4 mRNA expression. CONCLUSION: Induction of AdipoR gene expression in human skeletal muscle cells is not involved in the insulin-sensitizing effects of thiazolidinediones or fibrates.     

ROMK1 channel activity is regulated by monoubiquitination

The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.     

The melatonin receptor subtype MT1 is expressed in human gallbladder epithelia

Based on the fact that human bile and, particularly gallbladder bile, contains high physiological levels of the antioxidant melatonin, the aim of this study was to investigate whether the melatonin receptor MT1 is present in human gallbladder. Expression and localization of MT1 was assessed by RT-PCR, Western blotting and immunofluorescence analysis in gallbladder samples from patients with cholelithiasis and with advanced gallbladder carcinoma. Additionally, we monitored mRNA expression of the two key enzymes of melatonin synthesis, i.e. arylalkylamine-N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT). MT1 mRNA and protein were present in all cholelithiasis (n = 10) and gallbladder carcinoma (n = 5) samples. As indicated from RT-PCR and Western blot studies, MT1 is located in gallbladder epithelia. Epithelial expression was further proven by immunofluorescence staining of MT1 in paraffin-embedded cholelithiasis and gallbladder carcinoma sections. Analysis of AANAT and HIOMT mRNA expression showed that HIOMT mRNA is present in gallbladder. Surprisingly, AANAT was not detectable under conditions where it was found in a human colon specimen. The absence of AANAT suggests that in human gallbladder, HIOMT might be involved in the formation of 5-hydroxytryptamine products other than melatonin. In summary, our results provide the first evidence for the presence of MT1 in human gallbladder epithelia. Therefore, in addition to its profound antioxidative effects in the biliary system, melatonin might also act through MT1-mediated signal transduction pathways. Thereby, it might be involved in the regulation of gallbladder function.