Eric J. Romer

Hydrogen Peroxide Modulates Immunoglobulin Expression by Targeting the 3’Igh Regulatory Region through an NFκB-Dependent Mechanism

Abstract Reactive oxygen species such as hydrogen peroxide (H2O2) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H2O2 on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3′Igh regulatory region (3′IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H2O2 can enhance 3′IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H2O2 or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H2O2 concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H2O2 can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3′IghRR activity. These results may have significant implications to disease states previously associated with the 3′IghRR.

Protein Phosphatase 2A and Cdc7 Kinase Regulate the DNA Unwinding Element-binding Protein in Replication Initiation

Background: Cdc7 kinase and protein phosphatase 2A are important in DNA replication initiation. Results: DNA unwinding element (DUE)-binding protein (DUE-B) is an essential replication factor that is regulated by Cdc7 and PP2A. Conclusion: The phosphorylation of DUE-B determines its ability to bind the minichromosome maintenance helicase and regulate Cdc45 chromatin loading. Significance: PP2A and Cdc7 regulate DUE-B mediated loading of Cdc45 to the prereplication complex. The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2–7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2–7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.

Transgenic Expression of Spi-C Impairs B Cell Development and Function by Affecting Genes Associated With BCR Signaling

Spi‐C is an Ets family transcription factor closely related to PU.1 and Spi‐B. Expression of Spi‐C is developmentally regulated in the B‐cell lineage, but its function remains unknown. To determine the function of Spi‐C in B‐cell development, we generated mice expressing a B‐cell‐specific Spi‐C transgene under the control of the IgH intronic enhancer. Spi‐C transgenic mice had 50% fewer B cells than wild‐type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre‐B or immature B cells from transgenic mice were dramatically reduced compared with those of wild type. Both nonspecific and Ag‐specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared with wild type. Spi‐C transgenic B cells proliferated poorly after stimulation by anti‐IgM or anti‐CD40 in vitro, although they responded normally to LPS stimulation. Using real‐time RT‐PCR, we found that several BCR signaling‐related mediators were downregulated at pre‐B‐cell and mature B‐cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi‐C can impair B‐cell development and function by affecting genes associated with BCR signaling.

Platelet-Activating Factor–Induced Reduction in Contact Hypersensitivity Responses Is Mediated by Mast Cells via Cyclooxygenase-2–Dependent Mechanisms

Platelet-activating factor (PAF) stimulates numerous cell types via activation of the G protein–coupled PAF receptor (PAFR). PAFR activation not only induces acute proinflammatory responses, but it also induces delayed systemic immunosuppressive effects by modulating host immunity. Although enzymatic synthesis and degradation of PAF are tightly regulated, oxidative stressors, such as UVB, chemotherapy, and cigarette smoke, can generate PAF and PAF-like molecules in an unregulated fashion via the oxidation of membrane phospholipids. Recent studies have demonstrated the relevance of the mast cell (MC) PAFR in PAFR-induced systemic immunosuppression. The current study was designed to determine the exact mechanisms and mediators involved in MC PAFR-mediated systemic immunosuppression. By using a contact hypersensitivity model, the MC PAFR was not only found to be necessary, but also sufficient to mediate the immunosuppressive effects of systemic PAF. Furthermore, activation of the MC PAFR induces MC-derived histamine and PGE2 release. Importantly, PAFR-mediated systemic immunosuppression was defective in mice that lacked MCs, or in MC-deficient mice transplanted with histidine decarboxylase– or cyclooxygenase-2–deficient MCs. Lastly, it was found that PGs could modulate MC migration to draining lymph nodes. These results support the hypothesis that MC PAFR activation promotes the immunosuppressive effects of PAF in part through histamine- and PGE2-dependent mechanisms.

Systemic Platelet-Activating Factor-Receptor Agonism Enhances Non-Melanoma Skin Cancer Growth

Platelet-activating factor-receptor (PAF-R) agonists are pleiotropic lipid factors that influence multiple biological processes, including the induction and resolution of inflammation as well as immunosuppression. PAF-R agonists have been shown to modulate tumorigenesis and/or tumor growth in various skin cancer models by suppressing either cutaneous inflammation and/or anti-tumoral adaptive immunity. We have previously shown that a chronic systemic PAF-R agonist administration of mice enhances the growth of subcutaneously implanted melanoma tumors. Conversely, chronic topical applications of a PAF-R agonist suppressed non-melanoma skin cancer (NMSC) in a topical chemical carcinogenesis model (dimethylbenz[a]anthracene/phorbol 12-myristate 13-acetate (DMBA/PMA)) in-part via anti-inflammatory effects. These results indicate that the context of PAF-R agonist exposure via either chronic cutaneous or systemic administration, result in seemingly disparate effects on tumor promotion. To further dissect the contextual role of PAF-R agonism on tumorigenesis, we chronically administered systemic PAF-R agonist, carbamoyl-PAF (CPAF) to mice under a cutaneous chemical carcinogenesis protocol, recently characterized to initiate both NMSC and melanocytic nevus formation that can progress to malignant melanoma. Our results showed that while systemic CPAF did not modulate melanocytic nevus formation, it enhanced the growth of NMSC tumors.