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Cholecystokinin (CCK) and related peptides are potent growth factors in the gastrointestinal tract and may be important for human cancer. CCK exerts its growth modulatory effects through G(q)-coupled receptors (CCK(A) and CCK(B)) and activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). In the present study, we investigated the different mechanisms participating in CCK-induced activation of ERK1/2 in pancreatic AR42J cells expressing both CCK(A) and CCK(B). CCK activated ERK1/2 and Raf-1 to a similar extent as epidermal growth factor (EGF). Inhibition of EGF receptor (EGFR) tyrosine kinase or expression of dominant-negative Ras reduced CCK-induced ERK1/2 activation, indicating participation of the EGFR and Ras in CCK-induced ERK1/2 activation. However, compared with EGF, CCK caused only small increases in tyrosine phosphorylation of the EGFR and Shc, Shc-Grb2 complex formation, and Ras activation. Signal amplification between Ras and Raf in a CCK-induced ERK cascade appears to be mediated by activation of protein kinase Cepsilon (PKCepsilon), because 1) down-modulation of phorbol ester-sensitive PKCs inhibited CCK-induced activation of Ras, Raf, and ERK1/2 without influencing Shc-Grb2 complex formation; 2) PKCepsilon, but not PKCalpha or PKCdelta, was detectable in Raf-1 immunoprecipitates, although CCK activated all three PKC isoenzymes. In addition, the present study provides evidence that the Src family tyrosine kinase Yes is activated by CCK and mediates CCK-induced tyrosine phosphorylation of Shc. Furthermore, we show that CCK-induced activation of the EGFR and Yes is achieved through the CCK(B) receptor. Together, our data show that different signals emanating from the CCK receptors mediate ERK1/2 activation; activation of Yes and the EGFR mediate Shc-Grb2 recruitment, and activation of PKC, most likely PKCepsilon, augments CCK-stimulated ERK1/2 activation at the Ras/Raf level.
In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.
Erratum in: Correction: Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells. Journal of biological chemistry 2020 Oct 23;295(43):14792. doi: 10.1074/jbc.AAC120.016177.
Erratum for: Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor, thereby modulating activation of MAP kinase, Akt, and neurite outgrowth in PC12 cells.Journal of biological chemistry, 2002 Nov 15;277(46):43623-30. doi: 10.1074/jbc.M203926200. Epub 2002 Sep 5.
Aim of antiviral therapy of patients with chronic hepatitis C is the sustained elimination of the hepatitis C virus (HCV). The standard of care (SOC) is peginterferon alfa-2a/-2b with ribavirin for 48 weeks or 24 weeks in patients infected with HCV genotype 1 or 2/3, respectively. Overall, approximately half of the patients can be cured by SOC. Based on baseline viral load and the speed of virologic response during treatment, individualization of treatment duration is possible. However, this approach is not sufficient to substantially improve the sustained virologic response (SVR) rates. This goal can be achieved with new HCV specific inhibitors against the NS3/4A polymerase and the NS5B polymerase. Recent trials reported SVR rates in the order of 67-69% and 67-75% for the combination of SOC with the protease inhibitors telaprevir and boceprevir, respectively, in patients with HCV genotype 1 infection. Several new HCV specific inhibitors such as protease inhibitors, nucleoside and non-nucleoside polymerase inhibitors as well as non HCV specific compounds with anti-HCV activity such as cyclophilin inhibitors, silibinin, and nitazoxanide are currently in clinical evaluation. The review describes recent developments and discusses limitations posed by resistance development and drug toxicity.
Highly promising preclinical data obtained in cultured cells and in nude mice bearing xenografts contrast with the rather modest clinical efficacy of Polo-like kinase 1 (Plk1) inhibitors. In the present study, we investigated if Plk1 might be a suitable target in hepatocellular carcinoma (HCC) and if a genetically engineered mouse tumor model that well reflects the tumor cell and micro-environmental features of naturally occurring cancers might be suitable to study anti-Plk1 therapy. Analysis of Plk1 expression in human HCC samples confirmed that HCC express much higher Plk1 levels than the adjacent normal liver tissue. Inhibition of Plk1 by an adenovirus encoding for a short hairpin RNA against Plk1 or by the small-molecule inhibitor BI 2536 reduced the viability of HCC cell lines and inhibited HCC xenograft progression in nude mice. Treatment of transforming growth factor (TGF) α/c-myc bitransgenic mice with BI 2536 during hepatocarcinogenesis reduced the number of dysplastic foci and of Ki-67-positive cells within the foci, indicating diminished tumorigenesis. In contrast, BI 2536 had no significant effect on HCC progression in the transgenic mouse HCC model as revealed by magnetic resonance imaging. Measurement of BI 2536 by mass spectrometry revealed considerably lower BI 2536 levels in HCC compared with the adjacent normal liver tissue. In conclusion, low intratumoral levels are a novel mechanism of resistance to the Plk1 inhibitor BI 2536. Plk1 inhibitors achieving sufficient intratumoral levels are highly promising in HCC treatment.
Background & Aims: Thrombopoietin receptor agonists are a new class of compounds licenced for the treatment of immune thrombocytopenic purpura. They are currently being studied for patients with thrombopenia in advanced liver disease or under therapy for hepatitis C. There are indications that the risk for development of portal vein thrombosis in patients with advanced liver cirrhosis might be increased under therapy with thrombopoietin receptor agonists. We report a case of a patient with Child class B liver cirrhosis with concurrent immune thrombocytopenic purpura that developed portal vein thrombosis under therapy with the thrombopoietin receptor agonist romiplostim.
Methods: A 50-year-old woman with hepatitis C virus associated immune thrombocytopenic purpura and Child class B liver cirrhosis presented in our emergency with rapidly evolving hydropic decompensation and general malaise. For immune thrombocytopenic purpura, the patient was started on the thrombopoietin receptor agonist romiplostim nine months ago.
Results: During hospitalization, the platelet count was measured above 330,000/μl and partial portal vein thrombosis was diagnosed by imaging studies. The thrombotic event was assumed to be associated with the romiplostim treatment for immune thrombocytopenic purpura via excessive elevation of platelet count. After anticoagulation with heparin and cessation of romiplostim treatment, complete recanalisation of the portal vein was achieved.
Conclusions: We conclude that romiplostim should be used with precaution in patients with hepatitis C-associated immune thrombocytopenic purpura and advanced liver cirrhosis as the risk for thrombotic complications may increase significantly.
Background: Sphingolipids constitute bioactive molecules with functional implications in liver homeostasis. Particularly, ablation of very long chain ceramides in a knockout mouse model has been shown to cause a severe hepatopathy.
Methods: We aimed to evaluate the serum sphingolipid profile of 244 patients with cirrhosis prospectively followed for a median period of 228±217 days via mass spectrometry.
Results: We thereby observed a significant decrease of long and very long chain ceramides, particularly of C24ceramide, in patients with increasing severity of cirrhosis (p<0.001). Additionally, hydropic decompensation, defined by clinical presentation of ascites formation, was significantly correlated to low C24ceramide levels (p<0.001) while a significant association to hepatic decompensation and poor overall survival was observed for low serum concentrations of C24ceramide (p<0.001) as well. Multivariate analysis further identified low serum C24ceramide to be independently associated to overall survival (standard beta = -0.001, p = 0.022).
Conclusions: In our current analysis serum levels of very long chain ceramides show a significant reciprocal correlation to disease severity and hepatic decompensation and are independently associated with overall survival in patients with cirrhosis. Serum sphingolipid metabolites and particularly C24ceramide may constitute novel molecular targets of disease severity, hepatic decompensation and overall prognosis in cirrhosis and should be further evaluated in basic research studies.
Background & aims: Vitamin D, best known to regulate bone mineralization, has numerous additional roles including regulation inflammatory pathways. Recently, an increased incidence of 25-hydroxyvitamin D3 (25(OH)D3) deficiency has been found in subjects suffering from liver diseases. We here investigated if low vitamin D levels might be associated with prognosis, inflammation and infectious complications in patients with cirrhosis.
Methods: We performed a prospective cohort study investigating the relation between 25(OH)D3 levels and stages of cirrhosis, mortality and complications of cirrhosis, including infections.
Results: 251 patients with cirrhosis were enrolled into the present prospective cohort study. 25(OH)D3 levels were quantified by radioimmunoassay from serum samples obtained at study inclusion. The mean follow-up time was 411 ± 397 days with a range of 1-1382 days. 30 (12.0%) patients underwent liver transplantation and 85 (33.8%) individuals died within the study. The mean serum 25(OH)D3 concentration was 8.93 ± 7.1 ng/ml with a range of 1.0 to 46.0 ng/ml. 25(OH)D3 levels differed significantly between Child Pugh scores and showed a negative correlation with the model of end stage liver disease (MELD) score. Patients with decompensated cirrhosis and infectious complications, had significantly lower 25(OH)D3 levels compared to subjects without complications. Low 25(OH)D3 was associated with mortality in uni- as well as multivariate Cox regression models.
Conclusions: 25(OH)D3 deficiency is associated with advanced liver disease and low 25(OH)D3 levels are an indicator for a poor outcome and are associated with infectious complications.
Background: Autotaxin (ATX) and its product lysophosphatidic acid (LPA) are considered to be involved in the development of liver fibrosis and elevated levels of serum ATX have been found in patients with hepatitis C virus associated liver fibrosis. However, the clinical role of systemic ATX in the stages of liver cirrhosis was unknown. Here we investigated the relation of ATX serum levels and severity of cirrhosis as well as prognosis of cirrhotic patients.
Methods: Patients with liver cirrhosis were prospectively enrolled and followed until death, liver transplantation or last contact. Blood samples drawn at the day of inclusion in the study were assessed for ATX content by an enzyme-linked immunosorbent assay. ATX levels were correlated with the stage as well as complications of cirrhosis. The prognostic value of ATX was investigated by uni- and multivariate Cox regression analyses. LPA concentration was determined by liquid chromatography-tandem mass spectrometry.
Results: 270 patients were enrolled. Subjects with liver cirrhosis showed elevated serum levels of ATX as compared to healthy subjects (0.814±0.42 mg/l vs. 0.258±0.40 mg/l, P<0.001). Serum ATX levels correlated with the Child-Pugh stage and the MELD (model of end stage liver disease) score and LPA levels (r = 0.493, P = 0.027). Patients with hepatic encephalopathy (P = 0.006), esophageal varices (P = 0.002) and portal hypertensive gastropathy (P = 0.008) had higher ATX levels than patients without these complications. Low ATX levels were a parameter independently associated with longer overall survival (hazard ratio 0.575, 95% confidence interval 0.365–0.905, P = 0.017).
Conclusion: Serum ATX is an indicator for the severity of liver disease and the prognosis of cirrhotic patients.
Background: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.
Methods: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied.
Results: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles.
Conclusions: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.