Expression of laminin and its receptor LBP-32 in human and rat hepatoma cells.

Dramatic cellular changes that occur during hepatocarcinogenesis are associated with major alterations in extracellular matrix formation and in the relationships between cells and their microenvironment. We have studied the expression of laminin, the major noncollagenous glycoprotein of basement membrane, and the laminin receptor 32 kD laminin-binding protein in two rat (Faza 967 and HTC) and two human (HepG2 and HBGC2) hepatoma cell lines that express a variety of liver-specific functions. Laminin was found in the rough endoplasmic reticulum of these cells when the indirect immunoperoxidase method and electron microscopic examination were used. Radiolabeled laminin, immunoprecipitated from both media and cell extracts, was resolved by electrophoresis on sodium dodecyl sulfate gel in two major polypeptides that comigrated with the A and B subunits from Engelbreth-Holm-Swarm tumor laminin. Immunoblot analysis showed that the Mr = 400,000 polypeptide did not correspond to the A subunit of laminin. Northern blot analyses demonstrated large amounts of B1 and B2 mRNAs but no A chain mRNA. We conclude that the tumor cells produce the laminin B chains only. In contrast, normal adult hepatocytes from either man or rat lacked laminin mRNAs, whereas in 1-day primary culture, B chain mRNAs became detectable. The steady-state level of 32 kD laminin-binding protein mRNA was 10-fold and threefold higher in rat hepatoma cells than in freshly isolated and 1-day cultured normal rat hepatocytes, respectively. In human hepatocytes, the steady-state levels of 32 kD laminin-binding protein mRNAs varied depending on the donor and never reached the level of the human hepatoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of Dipeptidyl Peptidase-IV/CD26 and Aminopeptidase N/CD13 in Secretome of Mesenchymal Stem Cells after Treatment with LPS and PMA

Background: Emerging evidence suggests that secretome of mesenchymal stem cells has many anti-inflammatory and regenerative properties, which makes it a suitable candidate for the treatment of autoimmune and degenerative diseases. Dipeptidyl Peptidase-IV (DPP-IV)/CD26 and Aminopeptidase N (APN)/CD13 are ubiquitous ecto-enzymes which can digest various substrates including some chemokines and neuropeptides that are involved in inflammatory conditions. Objective: To evaluate the enzymatic activity of DPP-IV/CD26 and APN/CD13 in MSC conditioned media (MSC-CM). Methods: The MSCs were isolated from the mouse’s abdominal adipose tissues and were cultured without or with preconditioning by adding 2 µg/mL phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). The levels of interleukin-10 (IL-10), nitric oxide (NO), as well as the enzymatic activities of DPP-IV/CD26 and APN/CD13 were measured in MSC-CM. Results: The level of IL-10 and the enzyme activity of APN/CD13 did not show any changes in the MSC-CM of stimulated and non-stimulated cells. However, NO production was increased after treatment by LPS or PMA; nevertheless, the DPP-IV/CD26 activity was decreased in MSC-CM merely following the stimulation of cells with LPS. Conclusion: Our results indicated that MSC‐secretome had DPP-IV/CD26 and APN/CD13 activity. The DPP-IV/CD26 activity was decreased following stimulation of MSCs by toll-like receptor 4 agonist. Further studies are needed to reveal the possible contribution of DPP-IV/CD26 and APN/CD13 in the anti-inflammatory functions of MSC-CM.     

Multiple hormones regulate angiotensinogen messenger ribonucleic acid levels in a rat hepatoma cell line

Angiotensinogen is regulated by both hormones and changes in cardiovascular and electrolyte status. We have used the Reuber H35 (H4IIE) rat hepatoma cell line to study the regulation of angiotensinogen mRNA levels by dexamethasone, aldosterone, L-T3, and 17 beta-estradiol. Using the Acc I (1097 basepairs) fragment of our angiotensinogen cDNA clone, we have studied, by Northern and slot blot analysis, the accumulation of angiotensinogen mRNA in this cell culture system. Angiotensinogen mRNA of approximately 1800 bases was identified in these cells. It was identical in size to angiotensinogen mRNA from rat liver. Cells grown in medium containing serum depleted of thyroid and steroid hormones for up to 72 h showed a progressive decrease in angiotensinogen mRNA. Dexamethasone treatment resulted in a time- and dose-dependent increase in angiotensinogen mRNA. The half-maximal response occurred at 10(-9) M dexamethasone, with a maximal response of approximately 4-fold (serum-free conditions). Aldosterone induced a dose-dependent increase in mRNA. Half-maximal levels were obtained at 5 X 10(-8) M. Competition studies using the glucocorticoid antagonist RU38486 (Roussel-UCLAF) confirmed that dexamethasone was acting through the glucocorticoid receptor and suggested that aldosterone was acting through the same receptor. L-T3 treatment caused a dose and time-dependent increase in angiotensinogen mRNA levels. The half-maximal response occurred at 5 X 10(-8) M, and the maximal response was a 2-fold increase. Combined treatment with dexamethasone and L-T3 triiodothyronine resulted in a synergistic increase in angiotensinogen mRNA levels. 17 beta-Estradiol failed to elicit a change in angiotensinogen mRNA levels consistent with the observation that these cells lack estrogen receptors. Our results indicate that hepatic angiotensinogen mRNA levels are regulated in a complex fashion by several hormones. These cells provide a useful system for studying the hormonal regulation of the angiotensinogen gene.     

Polyamines and cytoskeletal proteins in intestinal epithelial cell migration

Abstract Our previous studies have shown that polyamines are essential for early mucosal restitution in vivo and cell migration in vitro. The current study determines whether cytoskeleton is involved in the process requiring polyamines for the stimulation of cell migration. Treatment with α-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, for 4 days totally inhibited ODC activity and depleted intracellular polyamines in the intestinal epithelial cells (IEC-6) derived from rat small intestinal crypt cells. Polyamine deficiency resulted in reorganization of F-actin in migrating cells but had no effect on the concentrations of filamentous actin and β-actin mRNA. The actin cortex was greatly increased in density and lamellipodia were less extensive. In contrast, non-muscle myosin I and II levels in DFMO-treated cells were decreased by 70 and 75%, respectively, and stress fibres were sparse or absent. The most striking feature of DFMO-treated cells was the appearance of many small punctate foci of myosin II in the cell interior. Migration of DFMO-treated cells was reduced by 80%. In the presence of DFMO, exogenous polyamine not only returned cytoskeleton levels and distribution towards normal but also restored cell migration to control levels. These results indicate that actin and myosins have a role in polyamine-dependent epithelial cell migration and may be part of the mechanism that requires polyamines for early mucosal restitution.     

Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells

Aims/hypothesis  Hepatic insulin degradation decreases in type 2 diabetes. Insulin-degrading enzyme (IDE) plays a key role in insulin degradation and its gene is located in a diabetes-associated chromosomal region. We hypothesised that IDE may be regulated by insulin and/or glucose in a liver cell model. To validate the observed regulation of IDE in vivo, we analysed biopsies of human adipose tissue during different clamp experiments in men. Methods  Human hepatoma HepG2 cells were incubated in normal (1 g/l) or high (4.5 g/l) glucose medium and treated with insulin for 24 h. Catalytic activity, mRNA and protein levels of IDE were assessed. IDE mRNA levels were measured in biopsies of human subcutaneous adipose tissue before and at 240 min of hyperinsulinaemic, euglycaemic and hyperglycaemic clamps. Results  In HepG2 cells, insulin increased IDE activity under normal glucose conditions with no change in IDE mRNA or protein levels. Under conditions of high glucose, insulin increased mRNA levels of IDE without changes in IDE activity. Both in normal and high glucose medium, insulin increased levels of the catalytically more active 15a IDE isoform compared with the 15b isoform. In subcutaneous adipose tissue, IDE mRNA levels were not significantly upregulated after euglycaemic or hyperglycaemic clamps. Conclusions/interpretation  Insulin increases IDE activity in HepG2 cells in normal but not in high glucose conditions. This disturbance cannot be explained by corresponding alterations in IDE protein levels or IDE splicing. The loss of insulin-induced regulation of IDE activity under hyperglycaemia may contribute to the reduced insulin extraction and peripheral hyperinsulinaemia in type 2 diabetes.     

Regulation of gene expression in rat hepatocytes and hepatoma cells by insulin: quantitation of messenger ribonucleic acid's coding for tyrosine aminotransferase, tryptophan oxygenase, and phosphoenolpyruvate carboxykinase

The effect of insulin on the abundance of mRNAs coding for tyrosine aminotransferase (TAT; EC 2.6.1.5), tryptophan oxygenase (TO; EC 1.13.1.12), and P-enolpyruvate carboxykinase(GTP) (PEPCK; EC 4.1.1.32) was examined in primary cultures of adult rat hepatocytes and in FTO-2B rat hepatoma cells by Northern blot analysis using RNA probes made from SP6-cDNAs. Insulin (10(-11)-10(-7) M), which has been reported to induce TAT and decrease the activity of TO, did not change the levels of TAT mRNA and TO mRNA in hepatocytes regardless of the presence of other inducers. In the same cells, dexamethasone increased TAT mRNA up to 19-fold and TO mRNA up to 15-fold, and 8pClPhS-cAMP (CPT-cAMP) raised the level of TAT mRNA up to 36-fold. The abundance of TO mRNA was not altered by CPT-cAMP. In contrast to TAT mRNA and TO mRNA, the level of PEPCK mRNA was dramatically decreased by insulin in the same hepatocytes. The sensitivity to this inhibitory effect of insulin was enhanced by dexamethasone and reduced by CPT-cAMP. FTO-2B hepatoma cells, which do not express detectable levels of TO mRNA, showed responses similar to those of hepatocytes, except that insulin caused a moderate reduction in TAT mRNA, but only in the presence of CPT-cAMP. The PEPCK mRNA in FTO-2B cells was suppressed by insulin in a manner closely resembling the effects in hepatocytes in the present study and in H4IIE hepatoma cells previously reported.     

Hepatic expression of CCAAT/enhancer binding protein α: hormonal and metabolic regulation in rats

Summary There is a significant body of evidence which suggests that the α-isoform of the CCAAT/enhancer binding protein (C/EBPα) plays a central regulatory role in energy metabolism in the liver. However, there is little information available regarding regulation of its expression in this tissue. In this study, we examined the effect of hormones and diabetes on its expression in rat H4IIE hepatoma cells and in rat liver. Treatment of H4IIE cells with dexamethasone led to a threefold increase in C/EBPα mRNA within 4 h. Insulin treatment produced a bi-phasic response, initially reducing mRNA levels up to the 4 h time point, but after 8 h a twofold increase in C/EBPα mRNA was observed. Treatment with 8-chlorophenylthio-cAMP produced a twofold induction of C/EBPα mRNA after 8 h. Western analysis indicated that the changes in mRNA in response to hormonal treatment generally resulted in corresponding alterations in C/EBPα protein levels. Finally, we observed an inhibition of C/EBPα gene expression in streptozotocin-diabetic rat liver, reflected by a decrease in both mRNA and protein levels that were partially reversed by insulin treatment. These results indicate that the expression of C/EBPα in liver is under complex control by both hormonal and metabolic signals, which is consistent with its role as a trans -regulator of genes which play a role in energy metabolism. [Diabetologia (1997) 40: 1117–1124] Received: 21 January 1997 and in revised form: 28 April 1997     

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.     

Effects of Ethanol on Rat Sertoli Cell Function: Studies In Vitro and In Vivo

Chronic alcohol administration to male animals is associated with testicular atrophy and gonadal failure. The Sertoli cell seems to be the first testicular cell injured as a result of alcohol exposure. To investigate the adverse effects of ethanol on testicular and particularly Sertoli cell function, the consequences of in vivo and in vitro ethanol exposure on rat Sertoli cell mRNA and protein levels of transferrin and ornithine decarboxylase were investigated. In vivo, ethanol exposure enhanced the levels of both hepatic and testicular (Sertoli cell) transferrin protein and mRNA. Ethanol exposure also enhanced testicular, but not hepatic, levels of ornithine decarboxylase protein and mRNA. These in vivo findings were confirmed when isolated Sertoli cells were studied in vitro. Specifically, ethanol exposure increased Sertoli cell transferrin protein and mRNA levels. Ethanol exposure increased Sertoli cell ornithine decarboxylase mRNA and protein when cultured in serum-free media, but not when cultured in the presence of serum. These studies demonstrate that ethanol exposure of rat Sertoli cells is associated with alterations in the levels of mRNA and protein that are known to be important in the process of spermatogenesis. These findings add to the body of evidence that suggests that, within the testes, the Sertoli cell may be an important target for ethanol-induced gonadal injury.     

二甲双胍对糖尿病大鼠心肌病理变化及凋亡相关基因的影响

目的观察二甲双胍对2型糖尿病大鼠心肌病理变化及心肌细胞中survivin、p27、c-myc及c-fos mRNA和蛋白变化的影响。方法建立糖尿病大鼠模型,35只糖尿病大鼠随机分为糖尿病组和二甲双胍组,另选15只大鼠作为正常对照组。喂养6周后,利用光镜和病理染色观察心肌病理改变,用RT-PCR检测心肌survivin、p27、c-myc及c-fos mRNA表达,用Western blot检测心肌survivin、p27、c-myc及c-fos蛋白表达。结果正常对照组心肌纤维排列整齐,心肌间质及血管周围间质成分少;糖尿病组心肌细胞肥大、水肿,心肌纤维排列紊乱,细胞间隙增宽,心肌间质成分明显增多,血管周围基质明显增多,可见较多炎性细胞浸润;二甲双胍组心肌纤维排列整齐,细胞间隙较窄,心肌间质和血管周围基质较少,少量炎性细胞浸润。二甲双胍组c-fos mRNA和蛋白的表达与正常对照组比较无显著性差异,但明显高于糖尿病组(P<0.05);二甲双胍组p27 mRNA和蛋白的表达明显低于正常对照组(P<0.05),且与糖尿病组比较无显著性差异(P>0.05);二甲双胍组c-myc mRNA和蛋白的表达与正常对照组和糖尿病组比较无显著性差异(P>0.05);二甲双胍组survivin mRNA和蛋白的表达低于正常对照组,高于糖尿病组,但无显著性差异(P>0.05)。结论糖尿病大鼠心肌病理改变与survivin、p27、c-fos mRNA及蛋白表达有关。二甲双胍对糖尿病心肌具有保护作用,其分子机制可能与改变心肌c-fos mRNA及蛋白的表达有关,而可能与survivin、p27、c-myc mRNA及其蛋白表达水平无关。     

Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: effects of warfarin

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.     

Decreased Ras-mitogen-activated protein kinase signaling may cause DNA hypomethylation in T lymphocytes from lupus patients

OBJECTIVE: Previous studies have shown that inhibiting T cell DNA methylation causes a lupus-like disease by modifying gene expression. T cells from patients with lupus exhibit diminished levels of DNA methyltransferase (MTase) enzyme activity, hypomethylated DNA, and changes in gene expression similar to those exhibited by T cells treated with methylation inhibitors, suggesting that DNA hypomethylation may contribute to human lupus. Since it is known that DNA MTase levels are regulated by the ras-mitogen-activated protein kinase (MAPK) pathway, this study sought to determine whether decreased ras-MAPK signaling could account for the DNA hypomethylation in lupus T cells. METHODS: DNA MTase messenger RNA (mRNA) from lupus patients and from healthy controls was quantitated by Northern analysis, and ras-MAPK signaling was determined by immunoblotting with antibodies to the activated forms of extracellular receptor-associated kinase (ERK). Results were compared with those in T cells in which ras-MAPK signaling was inhibited with a soluble inhibitor of MAPK ERK I (MEK1). RESULTS: T cells from patients with active lupus had diminished DNA MTase mRNA levels and decreased signaling through the ras-MAPK pathway. Inhibiting signaling through the ras-MAPK pathway with the MEK1 inhibitor decreased DNA MTase mRNA and enzyme activity to the levels seen in lupus T cells, and resulted in DNA hypomethylation resembling that seen in lupus T cells. CONCLUSION: These results suggest that a decrease in signaling through the ras-MAPK pathway may be responsible for the decreased MTase activity and DNA hypomethylation in patients with lupus.