Lori R. Covey

Nucleolin Is a Second Component of the CD154 mRNA Stability Complex That Regulates mRNA Turnover in Activated T Cells

CD154 (CD40L) mRNA turnover is regulated in part at the posttranscriptional level by a protein complex (termed Complex I) that binds to a highly CU-rich region of the 3′UTR. Polypyrimidine tract-binding protein (PTB) has previously been identified as a major RNA-binding protein in Complex I. Nondenaturing gel filtration of total extract from Jurkat T cells demonstrated that the CD154 mRNA-binding activity migrates as a ∼200-kDa complex, indicating the presence of multiple complex-associated proteins. We have currently undertaken a biochemical approach to further characterize Complex I and observed that it segregates over DEAE-Sepharose into two subcomplexes (termed I-L and I-U). Furthermore, nucleolin was identified as a component of both subcomplexes and was shown that it is the major RNA-binding protein in I-U. To directly demonstrate the biological significance of Complex I binding to the CD154 transcript, cytoplasm from human Jurkat cells was fractionated over a sucrose gradient and the different cellular fractions subjected to immunoprecipitation with anti-PTB and anti-nucleolin Abs. RT-PCR of the immunoprecipitated products using CD154-specific primers clearly demonstrated that nucleolin and PTB are associated with CD154 mRNA in both the ribonucleoprotein and polysome fractions. These data strongly support a model whereby nucleolin and PTB are integral to the stability of CD154 mRNA and are components of the CD154 ribonucleoprotein particle associated with actively translating ribosomes.

A Complex Containing Polypyrimidine Tract-Binding Protein Is Involved in Regulating the Stability of CD40 Ligand (CD154) mRNA

CD40 ligand (CD154) expression has been shown to be regulated, in part, at the posttranscriptional level by a pathway of “regulated instability” of mRNA decay throughout a time course of T cell activation. This pathway is modulated at late times of activation by the binding of a stability complex (termed complex I) to a CU-rich region in the 3′ untranslated region of the CD154 message. We have undertaken experiments to extend these findings and to analyze the cis-acting elements and trans-acting factors involved in this regulation. We have previously shown that the minimal binding sequence for complex I is a 63 nt CU-rich motif. However, our current study shows that when this site was deleted additional complex binding was observed upstream and downstream of the minimal binding region. Only after deletion of an extended region (termed Δ1515) was complex binding completely abolished. Analysis of complex binding using competition experiments revealed that the three adjacent regions bound related but not identical complexes. However, all three sites appeared to have a 55-kDa protein as the RNA-binding protein. Deletion of the Δ1515 region resulted in reduced transcript stability as measured by both in vitro and in vivo decay assays. Finally, using Abs against known RNA-binding proteins, we identified the polypyrimidine tract-binding protein (or heterogeneous nuclear ribonucleoprotein I) as a candidate RNA-binding component of complex I.

CD40:CD40L interactions in X-linked and non-X-linked hyper-IgM syndromes.

Hyper-IgM (HIM) syndrome is a rare immunodeficiency characterized by low or absent IgG, IgA, and IgE with normal or elevated levels of IgM. This disorder can be acquired or familial with either X-linked or autosomal patterns of inheritance. The X-linked form of the disease is a consequence of mutations in the CD40 ligand (CD40L) gene that encodes a protein expressed primarily on activated CD4+ T cells. The cognate interaction between CD40L on T cells and CD40 on antigen-stimulated B cells, macrophage, and dendritic cells is critical for the development of a comprehensive immune response. The non-X-linked form of HIM syndrome is heterogeneous and appears in some cases to be a consequence of mutations in the AID gene which encodes a B cells specific protein required for class switch recombination, somatic mutation, and germinal center formation. However, mutations in other unidentified genes are clearly the basis of the disease in a subset of patients. In this article, we review the essential features of the X-linked and non-X-linked forms of HIM syndrome and discuss the critical role the CD40∶CD40L receptor-ligand pair play in the pathogenesis of these immune deficiencies.

Identification of a Complex that Binds to the CD154 3′ Untranslated Region: Implications for a Role in Message Stability During T Cell Activation

CD154 expression is regulated throughout a time course of CD3-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the “stability” phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and CD3-activated CD4+ T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the CD154 3′ untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with CD154 mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at ∼90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-CD3-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a CD154-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a CD154-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of CD154 expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3′ untranslated region.

Post-transcriptional regulation in Lymphocytes: The case of CD154

The control of mRNA decay is emerging as an important control point and a major contributor to gene expression in both immune and non-immune cells. The identification of protein factors and cis-acting elements responsible for transcript degradation has illuminated a comprehensive picture of precisely orchestrated events required to both regulate and establish the decay process. One gene that is highly regulated at the post-transcriptional level is CD40 ligand (CD154 or CD40L). CD154 on CD4+ T cells is tightly controlled by an interacting network of transcriptional and post-transcriptional processes that result in precise surface levels of protein throughout an extended time course of antigen stimulation. The activation-induced stabilization of the CD154 transcript by a polypyrimidine tract-binding protein (PTB)-complex is a key event that corresponds to the temporal expression of CD154. In this review, we discuss known and potential roles of major mRNA decay pathways in lymphocytes and focus on the unique post-transcriptional mechanisms leading to CD154 expression by activated CD4+ T cells.

A Polypyrimidine Tract-Binding Protein-Dependent Pathway of mRNA Stability Initiates with CpG Activation of Primary B Cells

The mRNA encoding CD154, a critical protein involved in both humoral and cell-mediated immune responses, is regulated at the posttranscriptional level by the binding of complex I, a polypyrimidine tract-binding (PTB) protein-containing complex, which acts to increase message stability at late times of activation. Our current work focuses on analyzing a similar complex in B cells, designated B-cpx I, which is increased in B cells activated by CpG engagement of the TLR9 receptor but not by activation through CD40. Expression profiling of transcripts from primary B cells identified 31 mRNA transcripts with elevated PTB binding upon activation. Two of these transcripts, Rab8A and cyclin D2, contained binding sites for B-cpx I in their 3′ untranslated regions (UTRs). Analysis of turnover of endogenous Rab8A transcript in B cells revealed that like CD154, the mRNA half-life increased following activation and insertion of the Rab8A B-cpx I binding site into a heterologous transcript led to a 3-fold increase in stability. Also, short hairpin RNA down-regulation of PTB resulted in a corresponding decrease in Rab8A mRNA half-life. Overall these data strongly support a novel pathway of mRNA turnover that is expressed both in T cells and B cells and depends on the formation of a PTB-containing stability complex in response to cellular activation.

A Transcriptional Defect Underlies B Lymphocyte Dysfunction in a Patient Diagnosed with Non-X-Linked Hyper-IgM Syndrome

To establish the underlying cause of hyper-IgM syndrome in one female patient, B cell function was examined in response to CD40- and IL-4-mediated pathways. When CD40-induced functional responses were measured in unfractionated B cells, CD80 up-regulation, de novo Cμ-Cγ recombination, and Iγ transcription were all found to be relatively unaffected. However, CD40- and IL-4-mediated CD23 up-regulation and VDJ-Cγ transcription were clearly diminished compared to control cells. IL-4-induced CD23 expression was measurably reduced in the CD20− population as well. These results suggested that the patient’s defect is positioned downstream of CD40 contact and affects both CD40− and IL-4 signal transduction pathways. Further analysis of B cell function in CD19+ B cells revealed a clear B cell defect with respect to Iγ and mature VDJ-Cγ transcription and IgG expression. However, under the same conditions Iε transcription was relatively normal. Partial restoration of B cell function occurred if PBMC or CD19+ B cells were cultured in vitro in the presence of CD154 plus IL-4. Because addition of IL-4 to cocultures containing activated T cells failed to induce B cells to undergo differentiation, the ability of the patient’s B cells to acquire a responsive phenotype correlated with receiving a sustained signal through CD40. These findings support a model in which the patient expresses an intrinsic defect that is manifested in the failure of specific genes to become transcriptionally active in response to either CD154 or IL-4 and results in a functionally unresponsive B cell phenotype.

In vivo post-transcriptional regulation of CD154 in mouse CD4+ T cells

Interactions between CD40 and its ligand CD154 are involved in the progression of both cell mediated and innate immunity. These interactions are brought about by the transient expression of CD154 on activated CD4+ T cells, which is regulated, in part, at the level of mRNA turnover. Here we have focused on analyzing the pattern of post‐transcriptional regulation in mouse CD4+ T cells in response to activation. Initial experiments identify a region of the murine CD154 mRNA that binds a polypyrimidine tract‐binding protein‐containing complex (mComplex I), which is activation‐dependent and binds to a single CU‐rich site within the 3′ uTR Subsequent findings demonstrate that in vivo polyclonal activation of T cells leads to a pattern of differential CD154 mRNA stability that is directly dependent on extent of activation. Furthermore, in vitro activation of antigen‐primed T cells shows that the CD154 mRNA half‐life increases relative to that of unprimed cells. Importantly, this is the first report demonstrating that the regulation of CD154 in vivo is connected to an activation‐induced program of mRNA decay and thus provides strong evidence for post‐transcriptional mechanisms having a physiological role in regulating CD154 expression during an ongoing immune response.

A polymorphic CD40 ligand (CD154) molecule mediates CD40-dependent signalling but interferes with the ability of soluble CD40 to functionally block CD154:CD40 interactions.

We report the characterization of a naturally occurring polymorphism in CD40 ligand (CD40L, CD154) expressed by activated T cells from a young female patient. This polymorphism encodes a nonconservative Gly → Arg substitution in amino acid 219 in the extracellular, CD40 binding domain of the molecule. Studies carried out with 293 epithelial cells ectopically expressing the polymorphic protein (CD154/G219R) revealed reduced levels of binding to different anti‐CD154 monoclonal antibodies (mAb) and CD40‐immunoglobulin (CD40‐Ig). However, recognition of the polymorphic and wild‐type CD154 molecules by a polyclonal antiserum was comparable, suggesting that the polymorphism affects the ability of the protein to interact with CD40 but does not significantly alter its surface expression. To determine if reduced cross‐linking of CD40 mediated decreased functional effects, three CD40‐dependent properties were measured. We found that pathways leading to the induction of surface CD23, CD80, and Iγ transcription were activated in response to CD154/G219R signalling. However, the decrease in affinity for CD40 by the mutated CD154 affected the ability of CD40‐Ig to efficiently interfere with the binding and effectively block induced CD80 expression. In contrast, we found that the 5c8 mAb, which recognized the polymorphic molecule to a similar extent as wild‐type CD154, effectively blocked the interaction between CD154/G219R and CD40 as measured by CD80 expression. These findings suggest that naturally occurring polymorphisms in the CD154 molecule may affect the ability of CD40‐mediated functions to be blocked by soluble CD40 or anti‐CD154 mAb in the therapeutic treatment of disease and graft rejection.

N-benzyladriamycin-14-valerate (AD 198) exhibits potent anti-tumor activity on TRAF3-deficient mouse B lymphoma and human multiple myeloma.

BackgroundTRAF3, a new tumor suppressor identified in human non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), induces PKCδ nuclear translocation in B cells. The present study aimed to evaluate the therapeutic potential of two PKCδ activators, N-Benzyladriamycin-14-valerate (AD 198) and ingenol-3-angelate (PEP005), on NHL and MM.MethodsIn vitro anti-tumor activities of AD 198 and PEP005 were determined using TRAF3-/- mouse B lymphoma and human patient-derived MM cell lines as model systems. In vivo therapeutic effects of AD 198 were assessed using NOD SCID mice transplanted with TRAF3-/- mouse B lymphoma cells. Biochemical studies were performed to investigate signaling mechanisms induced by AD 198 or PEP005, including subcellular translocation of PKCδ.ResultsWe found that AD 198 exhibited potent in vitro and in vivo anti-tumor activity on TRAF3-/- tumor B cells, while PEP005 displayed contradictory anti- or pro-tumor activities on different cell lines. Detailed mechanistic investigation revealed that AD 198 did not affect PKCδ nuclear translocation, but strikingly suppressed c-Myc expression and inhibited the phosphorylation of ERK, p38 and JNK in TRAF3-/- tumor B cells. In contrast, PEP005 activated multiple signaling pathways in these cells, including PKCδ, PKCα, PKCϵ, NF-κB1, ERK, JNK, and Akt. Additionally, AD198 also potently inhibited the proliferation/survival and suppressed c-Myc expression in TRAF3-sufficient mouse and human B lymphoma cell lines. Furthermore, we found that reconstitution of c-Myc expression conferred partial resistance to the anti-proliferative/apoptosis-inducing effects of AD198 in human MM cells.ConclusionsAD 198 and PEP005 have differential effects on malignant B cells through distinct biochemical mechanisms. Our findings uncovered a novel, PKCδ-independent mechanism of the anti-tumor effects of AD 198, and suggest that AD 198 has therapeutic potential for the treatment of NHL and MM involving TRAF3 inactivation or c-Myc up-regulation.