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Regulation of CD40 function by its isoforms generated through alternative splicing.

ABSTRACT: CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-kappa B, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is regulated differentially in activated macrophages and dendritic cells. Pre-CD40 RNA is spliced preferentially out to signal-transducible CD40 mRNA in the early stage of activation; half of the CD40 mRNA is replaced by the signal-nontransducible CD40 mRNAs in the later stages (24 h). Using IL-12 p40 gene expression as a reporter for CD40 signaling, we show that three of the alternative isoforms can disable signaling through CD40. The major alternative isoform lacks the membrane-associated endodomain and seems to reduce the amount of the signal-transducible form available on the cell surface. It would seem, therefore, that CD40 expression is controlled by posttranscriptional and posttranslational regulation through alternative splicing. Modulation of isoform expression may provide a mechanism by which cells regulate their susceptibility to CD40L signaling.

SUBMITTER: Tone M

PROVIDER: S-EPMC29329 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Regulation of CD40 function by its isoforms generated through alternative splicing.

Tone M M Tone Y Y Fairchild P J PJ Wykes M M Waldmann H H

Proceedings of the National Academy of Sciences of the United States of America 20010201 4

CD40 is a member of the tumor necrosis factor receptor superfamily. The interaction between CD40 and CD40 ligand (CD154) activates NF-kappa B, Jun N-terminal kinase, and Janus kinase/signal transducers and activators of transcription pathways and promotes B cell growth, differentiation, and survival as well as IL-12 production in macrophages and dendritic cells. We demonstrate here the existence of multiple isoforms of CD40 mRNA generated by alternative splicing and show that their expression is ...[more]

PMID: 11172023

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Project description:IntroductionThe receptor activator of nuclear factor-kB (NF-kB) (RANK)/receptor activator of NF-kB ligand (RANKL) axis emerges as a key regulator of breast cancer initiation, progression and metastasis. RANK receptor is a tumor necrosis superfamily member, which upon ligand binding transduces a variety of survival, proliferation, differentiation and migration signals. The majority of these intracellular cues merge through the NF-kB transcription machinery.MethodsTNFRSF11A (RANK) variants were identified and cloned in mammalian expression vectors. Their expression was analyzed using real time PCR on RNA from normal tissue, cell lines and breast cancer specimens. Western blot analysis and immunofluoresence stainings were used to study expression and localization of protein isoforms in a panel of breast cancer cell lines and in transfected 293T cells. Luciferase assays were employed to assess the contribution of each isoform alone or in combinations on NF-kB activation. Isoform effect on cell survival after doxorubicin treatment was analyzed through MTT assay. Wound healing and transwell assays were employed to evaluate the effect of TNFRSF11A isoforms on migration of MDA-MB-231 and 293T cells.ResultsWe report the identification of three novel TNFRSF11A (RANK) variants, named TNFRSF11A_Δ9, TNFRSF11A_Δ8,9 and TNFRSF11A_Δ7,8,9 which result from the alternative splicing of exons 7 to 9. Interestingly, variant TNFRSF11A_Δ7,8,9 was found to be upregulated in breast cancer cells lines and its expression inversely correlated with tumor grade and proliferation index. TNFRSF11A_Δ7,8,9 encodes a 40-45 kDa protein, we named RANK-c, which lacks the transmembrane domain and most of the intracellular part of the wild type receptor. Furthermore, we showed that RANK-c could act as a dominant negative regulator of RANK-dependent NF-kB activation, affecting cell survival after apoptosis induction. In addition, RANK-c suppresses cell migration and represses the tumorigenic properties of invasive breast carcinoma cells.ConclusionsIn this study, we provide evidence of a complex regulatory network of RANK receptor splice variants with a role in breast cancer. We identify that the RANK-c isoform is expressed in breast cancer samples and its expression reversely correlates with histological grade. Finally, isoform RANK-c seems to have the capacity to regulate signaling through wild type RANK and moreover to inhibit cell motility and migration of breast cancer cells.

Project description:BackgroundTranscription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2) is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG). While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail.Principal findingsIn the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination.ConclusionsAltogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence about the functional diversity of the alternative TCF4 protein isoforms.

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Regulation of CD40 function by its isoforms generated through alternative splicing.

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Regulation of CD40 function by its isoforms generated through alternative splicing.

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