Biomedical subjects

Alexander L Dent

Publications and source records attributed to Alexander L Dent.

11 recordsLinked to original sources

PubMed2006

Regulation of Th2 cytokine expression in NKT cells: unconventional use of Stat6, GATA-3, and NFAT2.

NKT cells are unique in that they can produce high levels of both Th1 and Th2 cytokines, yet little is known about how NKT cells control the transcription of Th2 cytokines. The expression of IL-4 by NKT cells is independent of the Th2-associated transcription factor Stat6. We have found that Stat6 is critical for the expression of IL-5, IL-10, and IL-13 by NKT cells. However, the Th2 cell-associated transcription factor GATA-3, normally induced by Stat6 activation, is expressed at low levels in NKT cells. CD4+ NKT cells are highly enriched for Th2 cytokine expression compared with CD4- NKT cells, and we searched for transcription factors that are up-regulated in CD4+ NKT cells that could control Th2 cytokine expression. We found that the NFAT family member NFAT2 is selectively increased in CD4+ NKT cells. We tested the roles of NFAT2 and also GATA-3 in Th2 cytokine expression by retrovirus-mediated gene transduction into NKT cells and nonpolarized conventional T cells. Expression of NFAT2 increased the expression of IL-4 in both NKT cells and conventional T cells, and NFAT2 activated IL-10 in conventional T cells but not in NKT cells. GATA-3 strongly activated IL-4, IL-5, and IL-13 expression in conventional T cells but had comparatively weak effects on these cytokines in NKT cells. Thus, NFAT2, GATA-3, and Stat6 have surprisingly different roles in NKT cells than in conventional T cells. We propose that one mechanism by which CD4+ NKT cells express IL-4 independent of Stat6 is via increased NFAT2 activity.

Animals↗

PubMed2006

Caspase function and the immune system.

Caspases are key effector components of apoptosis, the process of programmed cell death. However, a large body of recent work has shown that caspase activity is necessary for several nonapoptotic cellular functions, such as proliferation, differentiation, survival, and the regulation of cytokine expression. In this review, we concentrate on the nonapoptotic functions of caspases and, in particular, on nonapoptotic roles of caspases in the immune system. Because of the involvement of caspases in promoting cell death, as well as cell proliferation and other nonapoptotic functions, studies involving the inhibition of caspases in vivo must be interpreted with caution.

Animals↗

PubMed2005

A role for caspases in controlling IL-4 expression in T cells.

Although caspase activation is critical for T cell proliferation following activation, the role of caspases in T cell differentiation is unclear. In this study, we have examined the effect of inhibition of caspases on the process of Th1/Th2 differentiation. Naive CD4+ T cells activated under neutral differentiation conditions in the presence of the pan caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (Z-VAD) fluoromethylketone showed increased Th2 cell differentiation concomitant with an up-regulation of GATA-3. Z-VAD induced optimal Th2 differentiation when T cells were stimulated under strong primary activation conditions. Treatment of naive CD4+ T cells with Z-VAD under strong activation conditions led to a 6-fold increase in IL-4 mRNA compared with control-treated T cells. The Z-VAD-induced increase in IL-4 transcription occurred within 24 h of activation and was independent of Stat6. IFN-gamma mRNA expression was not affected by Z-VAD at the 24-h time point. Z-VAD did not augment IL-4 expression from a committed Th2 cell, suggesting that caspases regulate IL-4 expression specifically during primary T cell activation. Z-VAD did not augment IL-12-driven Th1 differentiation. Activation of T cells in the presence of Z-VAD led to a specific increase in the expression of the transcription factor c-fos. Lastly, retrovirus-mediated expression of the antiapoptotic protein Bcl-2 resulted in an enhancement of Th2 cytokine expression, suggesting that inhibition of caspase activation by Bcl-2 can also modulate IL-4 expression. These findings reveal a novel regulatory mechanism of cytokine expression by caspases, and may explain how signaling pathways that inhibit apoptosis tend to promote Th2 differentiation.

Amino Acid Chloromethyl Ketones↗

PubMed2005

Regulation of IL-10 gene expression in Th2 cells by Jun proteins.

IL-10 is a key regulatory cytokine produced by T lymphocytes. Although Th2 cells are a major source of IL-10, little is known about IL-10 gene regulation in Th2 cells. High levels of IL-10 mRNA transcription are induced in the Th2 clone D10 after PMA plus ionomycin (P/I) stimulation; however we found that the IL-10 promoter was not inducible by P/I in D10 cells. We therefore sought regulatory regions in the IL-10 gene that could promote P/I-activated transcription in Th2 cells. Two strong DNase I-hypersensitive sites (DHSSs) were identified in the IL-10 gene in mouse T cells, and conserved noncoding sequences (CNSs) between the mouse and human IL-10 genes were also identified. One IL-10 DHSS maps within or next to a highly conserved CNS region, CNS-3. The CNS-3 region contains an AP-1 site that binds JunB and c-Jun proteins specifically in Th2 cells and not in Th1 cells. The CNS-3 element activates transcription from the IL-10 promoter after P/I stimulation and is responsive to c-Jun and JunB. Retroviral mediated-expression of either c-Jun or JunB in primary T cells led to a large increase in IL-10 expression, and inhibition of AP-1 activity by a dominant negative form of c-Jun in primary T cells strongly repressed IL-10 expression. IFN-gamma was relatively unaffected by modulations in AP-1 activity. These data indicate that we have identified a novel regulatory element that can specifically activate transcription of the IL-10 gene in Th2 cells via the AP-1/Jun pathway.

Animals↗

PubMed2004

BCL-6 negatively regulates macrophage proliferation by suppressing autocrine IL-6 production.

The transcription repressor BCL-6 is known to play critical roles in B-cell lymphomagenesis, germinal center formation, and balanced Th1/Th2 differentiation. In macrophages, although BCL-6 has also been shown to regulate the expression of several chemokine genes, its function in other aspects of macrophage biology has not been studied. In addition, the precise role of BCL-6 in cell proliferation is poorly understood in general. Here we report that BCL-6(-/-) macrophages hyperproliferate due to an accelerated G(1)/S transition accompanied by increased cyclin D2 and c-myc and decreased expression of p27. Crucial to this enhanced proliferation is spontaneous interleukin 6 (IL-6) production and signal transducer and activator of transcription 3 (STAT3) activation in BCL-6(-/-) macrophages. In colony-forming assays, BCL- 6(-/-) bone marrow progenitor cells form spontaneous macrophage colonies that can be inhibited by anti-IL-6 antibodies. Gene expression studies demonstrate that BCL-6 binds to several sequence motifs scattered in the IL-6 locus and can repress IL-6 transcription both in 293T cells and in macrophages. In conclusion, our results indicate that BCL-6 negatively regulates proliferation of the monocytic/macrophage lineage by suppressing an autocrine IL-6/STAT3-mediated gene expression program. Our work also suggests that BCL-6 prevents abnormal Th2 differentiation by suppressing basal level IL-6 production in antigen-presenting cells (APCs).

Animals↗

PubMed2004

Transcriptional repressor BCL-6 immortalizes germinal center-like B cells in the absence of p53 function.

Chromosomal rearrangements in non-Hodgkin's B-cell lymphoma implicate BCL-6 as an oncogene, yet direct evidence for BCL-6 acting as an oncogene in B cells has been lacking. Here, we show that BCL-6 can immortalize primary B cells, but only in the absence of p53 tumor suppressor function. The expression of BCL-6 led to greatly increased B-cell proliferation, particularly in response to CD40 stimulation. Furthermore, BCL-6-infected p53-deficient B cells gave rise to immortalized cell lines that could be maintained by CD40 stimulation. We found that in primary mouse B cells, BCL-6 repressed expression of the Blimp-1, p27kip1, and cyclin D2 target genes. BCL-6 did not markedly repress the PDCD2 and BCL-XL target genes. The BCL-6 immortalized cell lines had a phenotype consistent with germinal center B cells, they expressed the germinal center-specific M17 gene, and a significant fraction of the cells stained positive with PNA. Our data indicate that BCL-6 may act to maintain B cells in a germinal center-like state, and repression of Blimp-1 by BCL-6 may be particularly crucial for stabilization of the germinal center phenotype. Our data also suggest that disruption of the p53 pathway may be crucial for the development of BCL-6-expressing B-cell lymphomas.

Animals↗

PubMed2003

Inhibition of Th2 differentiation and GATA-3 expression by BCL-6.

The B cell lymphoma (BCL)-6 transcriptional repressor protein is an important regulator of Th2 responses. Mice deficient in BCL-6 develop severe Th2-type inflammation that can develop even in the absence of IL-4 signaling. We have investigated the mechanism for how BCL-6 regulates Th2 cell differentiation and have found that IL-6 signaling can promote dramatically increased levels of Th2 differentiation in BCL-6(-/-) CD4 T cells compared with wild-type CD4 T cells. IL-6 can induce a low level of Th2 cytokine expression in BCL-6(-/-)STAT6(-/-) cells but not in STAT6(-/-) cells. Since the promoters for Th2 cytokines such as IL-4, IL-5, IL-10, and IL-13 do not contain consensus BCL-6 DNA binding sites, we investigated whether BCL-6 might regulate the GATA-3 transcription factor that activates the expression of multiple Th2 cytokines. Consistent with the idea that BCL-6 represses GATA-3 expression, we found that GATA-3 levels are up-regulated in BCL-6(-/-)STAT6(-/-) CD4 T cells compared with STAT6(-/-) CD4 T cells. Retrovirus-mediated expression of BCL-6 in BCL-6(-/-)STAT6(-/-) T cells as well as developing wild-type Th2 cells leads to a potent repression of IL-4 and IL-10 secretion. Retrovirus-mediated expression of BCL-6 in both BCL-6(-/-)STAT6(-/-) and wild-type T cells also leads to a significant decrease in GATA-3 protein levels. Surprisingly, BCL-6 does not appear to regulate GATA-3 mRNA levels and thus BCL-6 appears to regulate GATA-3 expression at a posttranscriptional level. Regulation of GATA-3 protein levels is likely a key mechanism for how BCL-6 regulates Th2 cytokine expression and Th2 differentiation independently of STAT6. These data also point to a novel regulatory mechanism for BCL-6 separate from transcriptional repression.

Animals↗

PubMed2002

Repression of AP-1 function: a mechanism for the regulation of Blimp-1 expression and B lymphocyte differentiation by the B cell lymphoma-6 protooncogene.

The B cell lymphoma-6 (BCL-6) transcriptional repressor protein is an important regulator of B cell differentiation and is strongly implicated in the development of B cell lymphoma. Expression of the Blimp-1 transcription factor, which is critical for promoting B cell differentiation into plasma cells, is repressed by BCL-6. We have investigated the mechanism for how BCL-6 represses Blimp-1 transcription, and have found that BCL-6 regulates the Blimp-1 promoter through a novel mechanism involving AP-1 elements. Specifically, BCL-6 is a potent repressor of transcriptional activity mediated by AP-1 factors. We found that the zinc-finger region of BCL-6 interacts with c-Jun, JunB, and JunD proteins but does not bind c-Fos or Fra-2 proteins. An estrogen receptor ligand binding domain fusion with the BCL-6 zinc finger domain can act as a estrogen-inducible dominant negative protein and increase AP-1 activity in BCL-6(+) cells but not in BCL-6(-) cells, indicating that endogenous BCL-6 represses AP-1 activity. Additionally, we have confirmed a specific interaction between c-Jun and the zinc finger domain of BCL-6 in vivo using a mammalian two-hybrid assay. Repression of AP-1 function by BCL-6 may be a key mechanism for how BCL-6 regulates gene expression to control inflammation, lymphocyte differentiation, and lymphomagenesis.

B-Lymphocytes↗

PubMed2002

The forkhead transcription factor AFX activates apoptosis by induction of the BCL-6 transcriptional repressor.

The activation of the AKT/protein kinase B kinases by mutation of the PTEN lipid phosphatase results in enhanced survival of a diversity of tumors. This resistance to apoptosis is partly accomplished by the inhibition of genetic programs induced by a subfamily of forkhead transcription factors including AFX. Here we describe an AFX-regulated pathway that appears to account for at least part of this apoptotic regulatory system. Cells induced to synthesize an active form of AFX die by activating the apoptotic death pathway. An analysis of genes regulated by AFX demonstrated that BCL-6, a transcriptional repressor, is up-regulated approximately 4-7-fold. An examination of the BCL-6 promoter demonstrated that AFX bound to specific target sites that could activate transcription. BCL-X(L), an anti-apoptotic protein, contains potential BCL-6 target sites in its promoter. An analysis of endogenous BCL-X(L) levels in AFX-expressing cells revealed enhanced down-regulation of the transcript ( approximately 1.3-1.7-fold) and protein, and BCL-6 directly binds to and suppresses the BCL-X(L) promoter. Finally, macrophages isolated from BCL-6-/- mice show enhanced survival in vitro. These results suggest that AFX regulates apoptosis in part by suppressing the levels of anti-apoptotic BCL-XL through the transcriptional repressor BCL-6.

Animals↗

PubMed2002

Regulation of gene expression by the proto-oncogene BCL-6.

The proto-oncogene BCL-6 encodes a transcriptional repressor protein that is expressed at high levels in germinal center B cells and lymphomas with a germinal center B cell phenotype. The BCL-6 gene is a frequent target of chromosomal translocations, micro-deletions, and point mutations in non-Hodgkin's lymphoma. Studies of BCL-6-deficient mice have revealed that BCL-6 is critical for normal lymphocyte differentiation and also that BCL-6 is a negative regulator of inflammation. Recent studies have shed light on how BCL-6 controls these processes by showing that BCL-6 regulates a broad spectrum of target genes. BCL-6 represses transcription of genes involved in lymphocyte activation, differentiation, proliferation, and migration. Although much progress has been made in understanding gene regulation by BCL-6, many important questions are unresolved.

Animals↗

PubMed2002

Th1 cells regulate hematopoietic progenitor cell homeostasis by production of oncostatin M.

Regulation of hematopoietic progenitor cell homeostasis is crucial for maintenance of innate immunity and the ability of the body to respond to injury and infection. In this report, we demonstrate that progenitor cell numbers and cycling status in vivo are dramatically increased in mice deficient in Stat6 and decreased in mice deficient in Stat4, targeted mutations which also alter T helper cell polarization. Experiments using mice that have T cell restricted transgenic expression of Stat4 or Stat6 or have been in vivo depleted of T cell subsets demonstrate that CD4(+) T cells regulate progenitor cell activity. Injection of the Th1 cytokine Oncostatin M but not other cytokines into Stat4-deficient mice recovers progenitor cell activity to wild-type levels. Thus, T helper cells actively regulate hematopoietic progenitor cell homeostasis.

Animals↗