B cell receptor (BCR)-mediated antigen recognition is thought to regulate B cell differentiation. BCR signal strength may also influence B cell fate decisions. Here, we used the Epstein-Barr virus protein LMP2A as a constitutively active BCR surrogate to study the contribution of BCR signal strength in B cell differentiation. Mice carrying a targeted replacement of Igh by LMP2A leading to high or low expression of the LMP2A protein developed B-1 or follicular and marginal zone B cells, respectively. These data indicate that BCR signal strength, rather than antigen specificity, determines mature B cell fate. Furthermore, spontaneous germinal centers developed in gut-associated lymphoid tissue of LMP2A mice, indicating that microbial antigens can promote germinal centers independently of BCR-mediated antigen recognition.

Germinal centers (GCs) represent the main sites for the generation of high-affinity, class-switched antibodies during T cell-dependent antibody responses. To study gene function specifically in GC B cells, we generated Cγ1-cre mice in which the expression of Cre recombinase is induced by transcription of the Ig γ1 constant region gene segment (Cγ1). In these mice, Cre-mediated recombination at the fas , Ig β, IgH , and Rosa26 loci occurred in GC B cells as early as 4 days after immunization with T cell-dependent antigens and involved >85% of GC B cells at the peak of the GC reaction. Less than 2% of IgM + B cells showed Cre-mediated recombination. These cells carried few Ig somatic mutations, expressed germ-line Cγ1- and activation-induced cytidine deaminase-specific transcripts and likely include GC B cell founders and/or plasma cell precursors. Cre-mediated recombination involved most IgG1, but also a fraction of IgG3-, IgG2a-, IgG2b-, and IgA-expressing GC and post-GC B cells. This result indicates that a GC B cell can transcribe more than one downstream C H gene before undergoing class switch recombination. The efficient induction of Cre expression in GC B cells makes the Cγ1-cre allele a powerful tool for the genetic analysis of these cells, as well as, in combination with a suitable marker for Cre-mediated recombination, the tracking of class-switched memory B and plasma cells in vivo . To expedite the genetic analysis of GC B cells, we have established Cγ1-cre F 1 embryonic stem cells, allowing further rounds of gene targeting and the cloning of compound mutants by tetraploid embryo complementation.

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Phosphatidylinositol-4-kinase III␣ (PI4KIII␣) is an essential host cell factor for hepatitis C virus (HCV)replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for smallmolecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIII␣ inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIII␣ inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4III␣-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4III␣ inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIII␣ as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIII␣, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIII␣ inhibitors for treatment of chronic HCV infection.

In several cell types, mostly of epithelial origin, activation of the cAMP pathway triggers DNA synthesis and cell division. Regulation of gene expression by cAMP involves phosphorylation by protein kinase A (PKA) and activation of cAMP-response element binding protein (CREB)/CRE modulator (CREM) transcription factors which bind DNA to CRE sites. On the other hand, several CREM isoforms are transcriptional repressors, such as the inducible cAMP early repressor (ICER) transcription factors, which are synthesized from an intronic promoter of the CREM gene. This study investigated the potential role of CREB/CREM transcription factors in the cAMP mitogenic pathway, using an experimental model of epithelial cells in primary culture, i.e. dog thyroid cells stimulated by thyroid-stimulating hormone (TSH). In response to TSH, CREB/CREM transcription factors were phosphorylated on the serine residue of the PKA consensus site. In addition, the synthesis of ICER mRNAs was strongly induced by TSH. This transient upregulation of ICER expression correlated with increased protein levels. It was restricted to the cAMP pathway, as neither epidermal growth factor nor 12-O-tetradecanoylphorbol 13-acetate (TPA), which are potent mitogens for dog thyroid cells, induced ICER expression. On the other hand, increased expression of ICER mRNAs was not detected in dog thyroids chronically stimulated by TSH in vivo. The requirement for CREB/CREM transcription factors in the mitogenic effect of TSH was assessed by transfecting expression vectors encoding CREM repressors into dog thyrocytes in order to interfere with CRE-mediated gene transcription. The ectopic expression of ICER Ig or CREM a isoforms inhibited DNA replication in dog thyrocytes stimulated by TSH. This inhibitory effect was dependent on the ability of CREM repressors to form dimers but did not involve their DNA-binding capacity. Together these results show that CREB/CREM transcription factors are tightly regulated, at the transcriptional and post-translational levels, by TSH in dog thyroid cells, and provide clear evidence that their activity is required for the cAMP-dependent proliferation of epithelial cells in primary culture. Moreover, the transient induction of ICER transcription factors during mitogenic stimulation by TSH raises questions about the role of these potent repressors of CRE-dependent transcription as timers of cellular proliferation.Keywords: cAMP; cell proliferation; cAMP-response element binding protein/ CRE modulator (CREB/ CREM); inducible cAMP early repressor (ICER); thyroid. cAMP is an important second messenger involved in many physiological processes. However, its role in mitogenic decisions has remained controversial for a long time. Activation of the cAMP pathway can either trigger or prevent cellular proliferation depending on the cell type [1]. In early studies on fibroblasts, cAMP was described as a powerful inhibitor of cell division. On the other hand, hormones that activate the cAMP pathway are potent mitogens in a variety of epithelial cell...