A p38 MAPK-MEF2C pathway regulates B-cell proliferation.

B lymphocytes are an integral part of the adaptive immune system. On antigen binding to the B-cell receptor (BCR), B cells rapidly proliferate and differentiate into antibody-secreting plasma cells. The p38 mitogen-activated protein kinase (MAPK) pathway functions downstream of the BCR to control cell proliferation, but the transcriptional effectors of this pathway in B cells have remained elusive. In the present study, we inactivated Mef2c exclusively in B cells by conditional gene targeting in mice. Loss of MEF2C function resulted in a reduced immune response to antigen, defective germinal center formation, and a severe defect in B-cell proliferation, and we show that MEF2C regulates proliferation in response to BCR stimulation via the p38 MAPK pathway. p38 directly phosphorylates MEF2C via three residues in the C-terminal transactivation domain, establishing MEF2C as a direct transcriptional effector of BCR signaling via p38 MAPK.

Biolistic transformation of Schistosoma mansoni with 5' flanking regions of two peptidase genes promotes tissue-specific expression.

The gene-regulatory elements controlling peptidase expression in Schistosoma mansoni are unknown. A genomic DNA library was constructed from which 5' flanking fragments of the cathepsins F (SmCF; 649 bp) and B2 (SmCB2; 810 bp) peptidase genes were isolated. These were cloned into a GFP-expression vector for biolistic transformation of schistosomes. Both fragments promoted expression of GFP that was localised in the gut for SmCF and tegument for SmCB2, consistent with previous immunochemical data. Promoter-deletion of the SmCF gene indicated the importance of one or more transcription factor binding sites in the first 169 bp for both GFP-expression and its tissue specificity.

SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni.

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.

Mef2c is activated directly by Ets transcription factors through an evolutionarily conserved endothelial cell-specific enhancer.

Members of the Myocyte Enhancer Factor 2 (MEF2) family of transcription factors play key roles in the development and differentiation of numerous cell types during mammalian development, including the vascular endothelium. Mef2c is expressed very early in the development of the endothelium, and genetic studies in mice have demonstrated that mef2c is required for vascular development. However, the transcriptional pathways involving MEF2C during endothelial cell development have not been defined. As a first step towards identifying the transcriptional factors upstream of MEF2C in the vascular endothelium, we screened for transcriptional enhancers from the mouse mef2c gene that regulate vascular expression in vivo. In this study, we identified a transcriptional enhancer from the mouse mef2c gene sufficient to direct expression to the vascular endothelium in transgenic embryos. This enhancer is active in endothelial cells within the developing vascular system from very early stages in vasculogenesis, and the enhancer remains robustly active in the vascular endothelium during embryogenesis and in adulthood. This mef2c endothelial cell enhancer contains four perfect consensus Ets transcription factor binding sites that are efficiently bound by Ets-1 protein in vitro and are required for enhancer function in transgenic embryos. Thus, these studies identify mef2c as a direct transcriptional target of Ets factors via an evolutionarily conserved transcriptional enhancer and establish a direct link between these two early regulators of vascular gene expression during endothelial cell development in vivo.