- Email: [email protected]
MECHANISMS OF TRANSLATIONAL CONTROL OF APOLIPOPROTEIN B MRNA: IDENTIFICATION OF A NOVEL, INSULIN-SENSITIVE 110 KDA RNA BINDING PROTEIN MEDIATING TRANSLATIONAL STIMULATION OF APOLIPOPROTEIN B MRNA VIA CIS-TRANS INTERACTIONS AT THE 5′ UTR
Poster Abstracts / Cardiovascular Pathology 13 (2004) S80–S138 cardioplegia prior to global ischemia results in different gene expression changes as compared to control hearts. Therefore, myocardial protection during heart surgery using cardioplegic solutions has to be reevalutated on the gene expression level. This is especially interesting for the long term effect of CA on myocardial function in heart transplants.
P347 TRANSCRIPTION FACTOR NF KAPPAB AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IN THE DEVELOPMENT OF HBV-RELATED HEPATOCELLULAR CARCINOMA. George G Chen, Bertrand CH Leung, Joey S Y Chan, Xu Hu, Joseph WY Lau, Paul BS Lai. Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong. Nuclear factor (NF) kappaB is an important transcription factor that is involved in the growth and development of various cells and tissues. VEGF is known to participate the development of human hepatocellular carcinoma (HCC). The present study examined the expression of VEGF and p65, a subunit of NF-kappa B, in HCC liver tissues and non-tumor liver tissues. The activity of NF-kappa B in the cell nuclei isolated from HCC liver tissues and non-tumor liver tissues was also measured. The level of VEGF in sera of HCC patients was determined. The result showed that the concentration of the nuclear NF-kappa B was much higher in tumor tissues than in non-tumor tissues. The score of p65 expression was significantly increased in tumor tissues, compared with non-tumor tissues. VEGF was detected in both tumor and non-tumor tissues and mainly located in the cytoplasm rather than the nucleus. However, it appeared that the nucleus from tumor tissues was more frequently associated with p65 than that from non-tumor tissues. Correlation and regrssion analysis revealed a positive correlation between the nuclear NF-kappa B and the cytoplasmic VEGF in either tumor or non-tumor tissues. It has been known that the level of VEGF can be regulated by NF-kappa B and that the increased NF-kappa B stimulates the expression of VEGF. It appears that both NF-kappa B and VEGF promote the proliferation and growth of HCC cells by disrupting the balance between pro-apoptotic and anti-apoptotic molecules, with a shift towards the latter.
P348 MECHANISMS OF TRANSLATIONAL CONTROL OF APOLIPOPROTEIN B MRNA: IDENTIFICATION OF A NOVEL, INSULIN-SENSITIVE 110 KDA RNA BINDING PROTEIN MEDIATING TRANSLATIONAL STIMULATION OF APOLIPOPROTEIN B MRNA VIA CIS-TRANS INTERACTIONS AT THE 50 UTR. Konstantinos Gus Sidiropoulos, Louisa Pontrelli, Khosrow Adeli. Hospital for Sick Children, Department of Laboratory Medicine and Pathology University of Toronto, Toronto, Ontario, Canada. Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels without altering transcription and cellular mRNA levels. Mechanisms of apoB mRNA translational control and insulin-mediated modulation are largely unknown. Recent studies of apoB untranslated regions in our laboratory revealed a potential important role for cis-trans interactions at the 50 and 30 UTR. Deletion constructs of the UTR regions of apoB revealed that the 50 UTR was necessary and sufficient for insulin to inhibit synthesis of apoB. Using denaturing electrophoretic mobility shift assay (EMSA), protein complexes were detected binding to the 50 and 30 UTRs. Three transacting proteins of 75, 45, and 30 kDa bound directly to the 30 UTR, and a 110 kDa protein interacted with the 50 UTR. Non-denaturing EMSA determined that insulin increased binding of large complexes to the 50 UTR. Binding specificity was determined by competition with both
S127
specific and non-specific competitors. Binding of the 110 kDa factor to the 50 UTR was inhibited by all competitor fragments of the 50 UTR. Using radiolabeled fragments, we determined that binding was highest to those containing 50UTR apoB hairpin elements. Furthermore, 50 UTRLuciferase deletion mutants demonstrated that these hairpin structures were vital for optimal mRNA translation. We hypothesize that secondary structure of the apoB 50 UTR is vital for binding of the 110 kDa protein. Insulin treatment decreased binding of the 110 kDa protein to the 50 UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 50UTR by 2-fold. Analysis of the 30 UTR showed no significant changes in binding of trans-acting factors. We thus propose the existence of an insulin-sensitive factor that binds to the 50 UTR and regulates apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin resistant states could alter binding of such cistrans interactions at the 50 UTR and potentially lead to alterations in the rate of apoB synthesis. This work was supported by an operating grant from NSERC to KA. KGS is a recipient of a HSC restracomp scholarship and an OGS scholarship.
P349 HUMAN UMBILICAL VEIN ENDOTHELIAL CELL (HUVEC) RESISTANCE TO APOPTOSIS IS MEDIATED BY ACTIVATION OF NF-KAPPAB. Andrea R King, Sarah J Rowe, Sheila E Francis, Moira KB Whyte, David C Crossman. Universisty of Sheffield, Sheffield, UK. The cellular mechanisms that controls resistance to apoptosis in endothelial cells (EC) are poorly understood. Using two models of EC apoptosis, serum withdrawal (SW) and TRAIL treatment, we have consistently observed a population of cells (40 – 60% SW and 30% TRAIL) that are resistant to these stimuli after 24 hours and continue to proliferate. We determined if resistance to SW and TRAIL was dependent on position of the cell in the cell cycle and whether this was mediated by the protein kinase Akt or the transcription factor NF-kappaB, both well known endothelial survival factors. Culturing cells in low serum for 48 hours induced quiescence. Reduced rates of mitosis were demonstrated by time lapse video microscopy (TLVM), HUVEC+20% serum = 51.75%, HUVEC + 2% serum = 5.06%. After 24 hour SW, quiescent HUVEC were not resistant to SW-induced apoptosis (64.81 ± 8.43%) compared to proliferative HUVEC (53.88 ± 7.13% apoptosis) as quantified by TLVM, n = 3. In contrast, quiescent HUVEC were found to be more resistant to TRAIL-induced apoptosis (19.8 ± 5.32%) compared to proliferative HUVEC (78.7 ± 6.76%) apoptosis at 24 hours assessed by TLVM, n = 3. These data suggest that susceptibility to apoptosis is not solely due to EC position in the cell cycle. By Western blotting, Akt was not expressed in EC at any time point following SW. In response to TRAIL treatment, phosphorylated Akt was expressed to the same extent in both quiescent and proliferative HUVEC. These data suggest that under the conditions studied, Akt is not acting as a survival factor. By electrophoretic mobility shift assay (EMSA), NF-kappaB is present in EC nuclear extracts only at 24 and 48 hours after SW. In TRAIL-treated cells, nuclear expression is also only observed at later time points after TRAIL treatment (6, 24 and 48 hours). The expression of NF-kappaB in the nucleus may identify the apoptosis resistant population. These data may be of importance in identifying the molecular characteristics of normal apoptosis-resistant endothelium and in explaining the programmed response to an inflammatory stimulus.
P347 TRANSCRIPTION FACTOR NF KAPPAB AND VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IN THE DEVELOPMENT OF HBV-RELATED HEPATOCELLULAR CARCINOMA. George G Chen, Bertrand CH Leung, Joey S Y Chan, Xu Hu, Joseph WY Lau, Paul BS Lai. Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong. Nuclear factor (NF) kappaB is an important transcription factor that is involved in the growth and development of various cells and tissues. VEGF is known to participate the development of human hepatocellular carcinoma (HCC). The present study examined the expression of VEGF and p65, a subunit of NF-kappa B, in HCC liver tissues and non-tumor liver tissues. The activity of NF-kappa B in the cell nuclei isolated from HCC liver tissues and non-tumor liver tissues was also measured. The level of VEGF in sera of HCC patients was determined. The result showed that the concentration of the nuclear NF-kappa B was much higher in tumor tissues than in non-tumor tissues. The score of p65 expression was significantly increased in tumor tissues, compared with non-tumor tissues. VEGF was detected in both tumor and non-tumor tissues and mainly located in the cytoplasm rather than the nucleus. However, it appeared that the nucleus from tumor tissues was more frequently associated with p65 than that from non-tumor tissues. Correlation and regrssion analysis revealed a positive correlation between the nuclear NF-kappa B and the cytoplasmic VEGF in either tumor or non-tumor tissues. It has been known that the level of VEGF can be regulated by NF-kappa B and that the increased NF-kappa B stimulates the expression of VEGF. It appears that both NF-kappa B and VEGF promote the proliferation and growth of HCC cells by disrupting the balance between pro-apoptotic and anti-apoptotic molecules, with a shift towards the latter.
P348 MECHANISMS OF TRANSLATIONAL CONTROL OF APOLIPOPROTEIN B MRNA: IDENTIFICATION OF A NOVEL, INSULIN-SENSITIVE 110 KDA RNA BINDING PROTEIN MEDIATING TRANSLATIONAL STIMULATION OF APOLIPOPROTEIN B MRNA VIA CIS-TRANS INTERACTIONS AT THE 50 UTR. Konstantinos Gus Sidiropoulos, Louisa Pontrelli, Khosrow Adeli. Hospital for Sick Children, Department of Laboratory Medicine and Pathology University of Toronto, Toronto, Ontario, Canada. Insulin has been shown to acutely regulate hepatic apolipoprotein B (apoB) secretion at both translational and post-translational levels without altering transcription and cellular mRNA levels. Mechanisms of apoB mRNA translational control and insulin-mediated modulation are largely unknown. Recent studies of apoB untranslated regions in our laboratory revealed a potential important role for cis-trans interactions at the 50 and 30 UTR. Deletion constructs of the UTR regions of apoB revealed that the 50 UTR was necessary and sufficient for insulin to inhibit synthesis of apoB. Using denaturing electrophoretic mobility shift assay (EMSA), protein complexes were detected binding to the 50 and 30 UTRs. Three transacting proteins of 75, 45, and 30 kDa bound directly to the 30 UTR, and a 110 kDa protein interacted with the 50 UTR. Non-denaturing EMSA determined that insulin increased binding of large complexes to the 50 UTR. Binding specificity was determined by competition with both
S127
specific and non-specific competitors. Binding of the 110 kDa factor to the 50 UTR was inhibited by all competitor fragments of the 50 UTR. Using radiolabeled fragments, we determined that binding was highest to those containing 50UTR apoB hairpin elements. Furthermore, 50 UTRLuciferase deletion mutants demonstrated that these hairpin structures were vital for optimal mRNA translation. We hypothesize that secondary structure of the apoB 50 UTR is vital for binding of the 110 kDa protein. Insulin treatment decreased binding of the 110 kDa protein to the 50 UTR as visualized by EMSA. Absence of insulin increased binding of this trans-acting factor to the 50UTR by 2-fold. Analysis of the 30 UTR showed no significant changes in binding of trans-acting factors. We thus propose the existence of an insulin-sensitive factor that binds to the 50 UTR and regulates apoB mRNA translation. Perturbations in hepatic insulin signaling as observed in insulin resistant states could alter binding of such cistrans interactions at the 50 UTR and potentially lead to alterations in the rate of apoB synthesis. This work was supported by an operating grant from NSERC to KA. KGS is a recipient of a HSC restracomp scholarship and an OGS scholarship.
P349 HUMAN UMBILICAL VEIN ENDOTHELIAL CELL (HUVEC) RESISTANCE TO APOPTOSIS IS MEDIATED BY ACTIVATION OF NF-KAPPAB. Andrea R King, Sarah J Rowe, Sheila E Francis, Moira KB Whyte, David C Crossman. Universisty of Sheffield, Sheffield, UK. The cellular mechanisms that controls resistance to apoptosis in endothelial cells (EC) are poorly understood. Using two models of EC apoptosis, serum withdrawal (SW) and TRAIL treatment, we have consistently observed a population of cells (40 – 60% SW and 30% TRAIL) that are resistant to these stimuli after 24 hours and continue to proliferate. We determined if resistance to SW and TRAIL was dependent on position of the cell in the cell cycle and whether this was mediated by the protein kinase Akt or the transcription factor NF-kappaB, both well known endothelial survival factors. Culturing cells in low serum for 48 hours induced quiescence. Reduced rates of mitosis were demonstrated by time lapse video microscopy (TLVM), HUVEC+20% serum = 51.75%, HUVEC + 2% serum = 5.06%. After 24 hour SW, quiescent HUVEC were not resistant to SW-induced apoptosis (64.81 ± 8.43%) compared to proliferative HUVEC (53.88 ± 7.13% apoptosis) as quantified by TLVM, n = 3. In contrast, quiescent HUVEC were found to be more resistant to TRAIL-induced apoptosis (19.8 ± 5.32%) compared to proliferative HUVEC (78.7 ± 6.76%) apoptosis at 24 hours assessed by TLVM, n = 3. These data suggest that susceptibility to apoptosis is not solely due to EC position in the cell cycle. By Western blotting, Akt was not expressed in EC at any time point following SW. In response to TRAIL treatment, phosphorylated Akt was expressed to the same extent in both quiescent and proliferative HUVEC. These data suggest that under the conditions studied, Akt is not acting as a survival factor. By electrophoretic mobility shift assay (EMSA), NF-kappaB is present in EC nuclear extracts only at 24 and 48 hours after SW. In TRAIL-treated cells, nuclear expression is also only observed at later time points after TRAIL treatment (6, 24 and 48 hours). The expression of NF-kappaB in the nucleus may identify the apoptosis resistant population. These data may be of importance in identifying the molecular characteristics of normal apoptosis-resistant endothelium and in explaining the programmed response to an inflammatory stimulus.