Faculty Bibliography

Nonsense-mediated RNA decay (NMD) has long been viewed as an important constitutive mechanism to rapidly eliminate mutated mRNAs. More recently, it has been appreciated that NMD also degrades multiple nonmutated transcripts and that NMD can be regulated by wide variety of cellular stresses. Many of the stresses that inhibit NMD, including cellular hypoxia and amino acid deprivation, are experienced in cells exposed to hostile microenvironments, and several NMD-targeted transcripts promote cellular adaptation in response to these environmental stresses. Because adaptation to the microenvironment is crucial in tumorigenesis, and because NMD targets many mutated tumor suppressor gene transcripts, the regulation of NMD may have particularly important implications in cancer. This review briefly outlines the mechanisms by which transcripts are identified and targeted by NMD and reviews the evidence showing that NMD is a regulated process that can dynamically alter gene expression. Although much of the focus in NMD research has been in identifying the proteins that play a role in NMD and identifying NMD-targeted transcripts, recent data about the potential functional significance of NMD regulation, including the stabilization of alternatively spliced mRNA isoforms, the validation of mRNAs as bona fide NMD targets, and the role of NMD in tumorigenesis, are explored. Mol Cancer Res; 8(3); 295-308

The clinical relevance of heparin-induced antibodies (HIA) in the absence of thrombocytopenia remains to be defined. The aims of this study were (i) to determine the prevalence of HIA in patients treated by dialysis, (ii) to determine the prevalence of thrombocytopenia and heparin-induced thrombocytopenia (HIT), and (iii) to test whether HIA are associated with adverse outcomes. Sera from 740 patients treated by hemodialysis (HD, n=596) and peritoneal dialysis (PD, n=144) were tested for HIA (IgG, IgA or IgM) by masked investigators at approximately six months after enrolment in the Choices for Healthy Outcomes in Caring for End-Stage Renal Disease (CHOICE) study. We assessed, with time-to-event Cox proportional hazards models, whether the presence of HIA predicted any of four clinical outcomes: arterial cardiovascular events, venous thromboembolism, vascular access occlusion and mortality. HIA prevalence was 10.3% overall. HIA positivity did not predict development of thrombocytopenia or any of the four clinical outcomes over a mean follow-up of 3.6 years, with hazard ratios for arterial cardiovascular events of 0.98 (95% confidence interval 0.70-1.37), venous thromboembolism 1.39 (0.17-11.5), vascular access occlusion 0.82 (0.40-1.71), and mortality 1.18 (0.85-1.64). Chronic intermittent heparin exposure was associated with a high seroprevalence of HIA. In dialysis patients these antibodies were not an independent risk factor for cardiovascular events and mortality. Our data do not suggest that dialysis patients should be monitored for HIA antibodies in the absence of thrombocytopenia

Many tumors are hypoxic, and cells that are experimentally rendered hypoxic display a variety of phenotypes which allow them to adapt to the micro-environment. These phenotypes include a shift from aerobic to anaerobic metabolism, a diminution of reactive oxygen species, an arrest of proliferation, apoptosis, and a secretion of pro-angiogenic growth factors. Some of these hypoxic phenotypes are re-capitulated in normoxic tumor cells (e.g., an increase in anaerobic metabolism), and some tumors have undergone mutations that allow them to bypass the cell cycle arrest and apoptosis typically seen in hypoxic cells. Hypoxic regulation of gene expression is responsible for many hypoxia-induced phenotypes, and here we review a variety of mechanisms by which gene expression is altered in hypoxic cells. These include transcription by HIF-1, the hypoxia inducible transcription factor, and other hypoxia-inducible transcription factors, including ones generated by hypoxic activation of the integrated stress response. Recent data from our laboratory demonstrate that nonsense mediated RNA decay is also regulated in hypoxic cells and thus may play an important role in hypoxic gene regulation and hypoxic phenotypes

Nonsense-mediated RNA decay (NMD) rapidly degrades both mutated mRNAs and nonmutated cellular mRNAs in what is thought to be a constitutive fashion. Here we demonstrate that NMD is inhibited in hypoxic cells and that this inhibition is dependent on phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). eIF2alpha phosphorylation is known to promote translational and transcriptional up-regulation of genes important for the cellular response to stress. We show that the mRNAs of several of these stress-induced genes are NMD targets and that the repression of NMD stabilizes these mRNAs, thus demonstrating that the inhibition of NMD augments the cellular stress response. Furthermore, hypoxia-induced formation of cytoplasmic stress granules is also dependent on eIF2alpha phosphorylation, and components of the NMD pathway are relocalized to these granules in hypoxic cells, providing a potential mechanism for the hypoxic inhibition of NMD. Our demonstration that NMD is inhibited in hypoxic cells reveals that the regulation of NMD can dynamically alter gene expression and also establishes a novel mechanism for hypoxic gene regulation

OBJECTIVE: Identification of isolated congenital heart block (CHB) predicts, with near certainty, the presence of maternal anti-SSA/Ro antibodies; however, the 2% incidence of CHB in first offspring of anti-SSA/Ro+ mothers, 20% recurrence in subsequent pregnancies, and discordance in identical twins suggest that an environmental factor amplifies the effect of the antibody. Accordingly, this study was carried out to explore the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal cardiac fibroblast. METHODS: Evidence of an effect of hypoxia was sought by immunohistologic evaluation of CHB-affected fetal heart tissue and by determination of erythropoietin levels in cord blood. The in vitro effect of hypoxia on gene expression and phenotype in fibroblasts derived from fetal hearts and lungs was investigated by Affymetrix arrays, quantitative polymerase chain reaction (PCR), immunofluorescence, and immunoblotting. RESULTS: In vivo hypoxic exposure was supported by the prominent intracellular fibroblast expression of hypoxia-inducible factor 1alpha in conduction tissue from 2 fetuses in whom CHB led to death. The possibility that hypoxia was sustained was suggested by significantly elevated erythropoietin levels in cord blood from CHB-affected, as compared with unaffected, anti-SSA/Ro-exposed neonates. In vitro exposure of cardiac fibroblasts to hypoxia resulted in transdifferentiation to myofibroblasts (a scarring phenotype), as demonstrated on immunoblots and immunofluorescence by increased expression of smooth muscle actin (SMA), an effect not seen in lung fibroblasts. Hypoxia-exposed cardiac fibroblasts expressed adrenomedullin at 4-fold increased levels, as determined by Affymetrix array, quantitative PCR, and immunofluorescence, thus focusing attention on cAMP as a modulator of fibrosis. MDL12,330A, an adenylate cyclase inhibitor that lowers the levels of cAMP, increased expression of fibrosis-related proteins (mammalian target of rapamycin, SMA, plasminogen activator inhibitor type 1, and type I collagen), while the cAMP activator forskolin attenuated transforming growth factor beta-elicited fibrosing end points in the cardiac fibroblasts. CONCLUSION: These findings provide evidence that hypoxia may amplify the injurious effects of anti-SSA/Ro antibodies. Modulation of cAMP may be a key component in the scarring phenotype. Further assessment of the susceptibility of cardiac fibroblasts to cAMP modulation offers a new research direction in CHB

The Id proteins play an important role in proliferation, differentiation and tumorigenesis. Many tumors are hypoxic, but it is unknown if expression of Id proteins is regulated in hypoxic cells. Here we show that Id-1 is down-regulated in multiple primary, immortalized, and neoplastic hypoxic cell lines, and the transcriptional repressor ATF-3 is both necessary and sufficient for this hypoxia-induced repression of Id-1. Hypoxic up-regulation of ATF-3 is due in part to activation of the unfolded protein response, a cellular stress response. Remarkably, we observe that the unfolded protein response is de-regulated in all neuroblastoma cell lines tested. Indeed, in the absence of ATF-3 the hypoxia-induced transcription factor HIF-1 up-regulates Id-1 in hypoxic neuroblastoma cells. Hypoxic neuroblastoma cells diminish expression of some neuronal differentiation markers, and forced expression of ATF-3 in hypoxic neuroblastoma cells represses Id-1 and prevents the loss of these markers. The divergent regulation of Id proteins in distinct hypoxic cells may explain some of the varied effects hypoxia has on cellular differentiation and proliferation