Inês Soeiro

Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle

Background. Mesenchymal stem cells (MSCs) play a crucial role in hematopoietic development and have been shown to exert a powerful immunosuppressive effect. In this study, we investigated the effect of bone marrow MSC on the differentiation and function of peripheral blood monocytes into dendritic cells (DCs). Methods. Human MSCs, generated from normal bone marrow, were added to peripheral blood monocytes stimulated in vitro with granulocyte–macrophage colony stimulating factor and interleukin-4 to become DCs. Monocytes were then examined for the expression of markers characteristic of DCs and their ability to stimulate allogeneic T cells. In addition, the effect of MSCs on the cell cycle of monocyte-derived DCs and the expression of various cell cycle proteins were analyzed by cytometric analysis and Western blotting with specific antibodies. Results. MSCs blocked the differentiation of monocytes into DCs and impaired their antigen-presenting ability. This resulted from a block of monocytes from entering the G1 phase of the cell cycle with a progressive number of cells accumulating in the G0 phase. Cyclin D2 was downregulated. However, differently from what was observed in T-cells stimulated in the presence of MSCs, the expression of p27kip1 was found decreased, suggesting the involvement of similar but not identical pathways. Conclusions. We conclude that MSCs impair monocyte differentiation and function by interfering with the cell cycle. These findings imply that MSC-induced immunosuppression might be a side product of a more general antiproliferative effect.

Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells

In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively phosphorylated in the Bcr-Abl-positive BV173 and BaF3/Bcr-Abl cells. Inhibition of Bcr-Abl kinase by STI571 results in FoxO3a activation, induction of Bim expression and apoptosis. Using reporter gene assays, we demonstrate that STI571 and FoxO3a activate Bim transcription through a FoxO-binding site (FHRE) located within the promoter. This was verified by DNA pull-down and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to induction of Bim expression and apoptosis. Conversely, silencing of FoxO3a in Bcr-Abl-expressing cells abolishes STI571-mediated Bim induction and apoptosis. Together, the results presented clearly confirm FoxO3a as a key regulator of apoptosis induced by STI571, and show that Bim is a direct transcriptional target of FoxO3a that mediates the STI571-induced apoptosis. Thus, STI571 induces an accumulation of FoxO3a activity which in turn binds directly to an FHRE in the promoter to activate Bim expression and apoptosis.

FoxO3a and BCR-ABL Regulate cyclin D2 Transcription through a STAT5/BCL6-Dependent Mechanism

ABSTRACT Cell cycle arrest by FoxO transcription factors involves transcriptional repression of cyclin D, although the exact mechanism remains unclear. In this study, we used the BCR-ABL-expressing cell line BV173 as a model system to investigate the mechanisms whereby FoxO3a regulates cyclin D2 expression. Inhibition of BCR-ABL by STI571 results in down-regulation of cyclin D2 expression, activation of FoxO3a activity, and up-regulation of BCL6 expression. Using reporter gene assays, we demonstrate that STI571, FoxO3a, and BCL6 can repress cyclin D2 transcription through a STAT5/BCL6 site located within the cyclin D2 promoter. We propose that BCR-ABL inhibition leads to FoxO3a activation, which in turn induces the expression of BCL6, culminating in the repression of cyclin D2 transcription through this STAT5/BCL6 site. This process was verified by mobility shift and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to accumulation of BCL6 and down-regulation of cyclin D2 at protein and mRNA levels. Furthermore, silencing of FoxO3a and BCL6 in BCR-ABL-expressing cells abolishes STI571-mediated effects on cyclin D2. This report establishes the signaling events whereby BCR-ABL signals are relayed to cyclin D2 to mediate cell cycle progression and defines a potential mechanism by which FoxO proteins regulate cyclin D2 expression.

BCR targets cyclin D2 via Btk and the p85alpha subunit of PI3-K to induce cell cycle progression in primary mouse B cells.

The p85α subunit of PI3-K and Btk are two crucial components of the B-cell receptor (BCR) signalling pathway. In the present study, we showed that primary splenic B cells from p85α null and xid (Btk-deficient) mice fail to induce cyclin D2 expression and enter early G1, but not S phase of the cell cycle in response to BCR engagement. Furthermore, these Btk or p85α null B cells displayed increased cell death compared with wild type following BCR engagement. These findings are further confirmed by studies showing that specific pharmacological inhibitors of Btk (LFM-A13), PI3-K (LY294002 and Wortmannin) and PLCγ (U73122) also block cyclin D2 expression and S phase entry following BCR stimulation, as well as triggering apoptosis. Collectively, these data provide evidence for the concept that the B-cell signalosome (p85α, Btk, BLNK and PLCγ) is involved in regulating cyclin D2 expression in response to BCR engagement. PKC and intracellular calcium are two major downstream effectors of the B-cell signalosome and can be activated by PMA and ionomycin, respectively. In small resting (G0) B cells, costimulation with PMA and ionomycin, but not PMA or ionomycin alone, induces cyclin D2 expression and cell-cycle progression. Consistent with this, we also showed that the BCR-mediated cyclin D2 induction could be abolished by pretreatment of resting B cells with specific inhibitors of capacitative Ca2+ entry (SK&F 96365) or PKC (Gö6850). Our present results lead us to propose a model in which the B-cell signalosome targets cyclin D2 via the Ca2+ and PKC-dependent signalling cascades to mediate cell-cycle progression in response to BCR engagement.

Murine γ-Herpesvirus 68 Latency Protein M2 Binds to Vav Signaling Proteins and Inhibits B-cell Receptor-induced Cell Cycle Arrest and Apoptosis in WEHI-231 B Cells

The MHV-68 latent protein, M2, does not have homology to any known viral or cellular proteins, and its function is unclear. To define the role played by M2 during MHV-68 latency as well as the molecular mechanism involved, we used M2 as bait to screen a yeast two-hybrid mouse B-cell cDNA library. Vav1 was identified as an M2-interacting protein in two independent screenings. Subsequent yeast two-hybrid interaction studies showed that M2 also binds to Vav2, but not Vav3, and that three “PXXP” motifs located at the C terminus of M2 are important for this interaction. The interactions between M2 and Vav proteins were also confirmed in vivo in 293T and WEHI-231 B-cells by co-immunoprecipitation assays. Rac1/GST-PAK “pull-down” experiments and Western blot analysis using a phospho-Vav antibody demonstrated that expression of M2 in WEHI-231 cells enhances Vav activity. We further showed in WEHI-231 cells that M2 expression promotes proliferation and survival and is associated with enhanced cyclin D2 and repressed p27Kip1, p130, and Bim expression. Taken together, these experiments suggest that M2 might have an important role in disseminating the latent virus during the establishment and maintenance of latency by modulating B-cell receptor-mediated signaling events through Vav to promote B-cell activation, proliferation, and survival.

Cyclin D2 controls B cell progenitor numbers

Cyclin D2 affects B cell proliferation and differentiation in vivo. It is rate‐limiting for B cell receptor (BCR)‐dependent proliferation of B cells, and cyclin D2−/− mice lack CD5+(B1) B lymphocytes. We show here that the bone marrow (BM) of cyclin D2−/− mice contains half the numbers of Sca1+B220+ B cell progenitors but normal levels of Sca1+ progenitor cells of other lineages. In addition, clonal analysis of BM from the cyclin D2−/− and cyclin D2+/+ mice confirmed that there were fewer B cell progenitors (B220+) in the cyclin D2−/− mice. In addition, the colonies from cyclin D2−/− mice were less mature (CD19lo) than those from cyclin D2+/+ mice (CD19Hi). The number of mature B2 B cells in vivo is the same in cyclin D2−/− and cyclin D2+/+ animals. Lack of cyclin D2 protein may be compensated by cyclin D3, as cyclin‐dependent kinase (cdk)6 coimmunoprecipitates with cyclin D3 but not cyclin D1 from BM mononuclear cells of cyclin D2−/− mice. It is active, as endogenous retinoblastoma protein is phosphorylated at the cdk6/4‐cyclin D‐specific sites, S807/811. We conclude that cyclin D2 is rate‐limiting for the production of B lymphoid progenitor cells whose proliferation does not depend on BCR signaling.

p27Kip1 and p130 Cooperate To Regulate Hematopoietic Cell Proliferation In Vivo

ABSTRACT To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1−/−; p130−/− mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1−/− counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1−/−; p130−/− animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1−/− splenocytes. The finding that the p27Kip1−/−; p130−/− splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

Phosphatidylinositol 3-kinase is required for the transcriptional activation of cyclin D2 in BCR activated primary mouse B lymphocytes.

Induction of cyclin D2 is essential for mediating cell cycle entry in B cells activated by BCR cross‐linking. In the present study we show that, like B lymphocytes lacking cyclin D2, the p85α subunit of phosphatidylinositol 3‐kinase (PI3K) or other components of the B cell signalosome, p110δ‐null B cells fail to induce cyclin D2 and enter early G1 but not S phase of the cell cycle. The inhibitors of PI3K activity, LY294002 and Wortmannin, also abrogate cyclin D2 induction by BCR cross‐linking, confirming that the class IA PI3K is necessary for cyclin D2 induction in response to BCR stimulation. Furthermore, using both p85α‐null and p110δ‐null B cells and inhibitors of PI3K, this study demonstrates for the first time, that BCR cross‐linking induces cyclin D2 mRNA expression via transcriptional activation of the cyclin D2 promoter and that this transcriptional activation of cyclin D2 requires PI3K activity. Moreover, we identify a region between nucleotides ‐1624 and ‐1303 of the cyclin D2 promoter containing elements responsive to anti‐IgM, which are PI3K dependent. Further characterisation of signalling intermediates downstream of the BCR revealed a perturbation of MAPK signalling pathways in p85α‐null and p110δ‐null B cells, and our data suggests that cross‐talk exists between the PI3K and JNK pathways.