Paritosh Ghosh

RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter.

T-cell activation requires two different signals. The T-cell receptors recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.

Effect of Promoter Methylation on the Regulation of IFN-γ Gene During In Vitro Differentiation of Human Peripheral Blood T Cells into a Th2 Population

The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by Th1 and Th2 cell subsets. Th1 cells preferentially produce IL-2 and IFN-γ, resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10, stimulating an Ab response that attacks extracellular pathogens, thereby preventing the cells from becoming infected. To elucidate the mechanisms of differential regulation of cytokine genes by these two different subsets of T cells, we established an in vitro differentiation model of freshly isolated human peripheral blood T cells in which IFN-γ was used as an index gene to study the transcriptional regulation. The data presented here demonstrate that the IFN-γ promoter undergoes differential methylation during in vitro differentiation: the promoter becomes hypermethylated in Th2 cells, whereas it is hypomethylated in Th1 cells. Hypermethylation in Th2 cells results in chromatin condensation and exclusion of CREB proteins from the IFN-γ promoter. Treatment with 5-azacytidine, a demethylating agent, causes Th2 cells to reverse histone condensation and enables CREB recruitment to the hypomethylated promoter. This results in the increased production of IFN-γ. These data indicate the importance of promoter methylation in the regulation of the IFN-γ gene during differentiation.

Kappa B site-dependent activation of the interleukin-2 receptor alpha-chain gene promoter by human c-Rel.

The cis-acting control elements of the interleukin-2 receptor alpha-chain (IL-2R alpha) gene contain a potent kappa B-like enhancer whose activity can be induced by various mitogenic stimuli. Recent cloning of the p50 and p65 subunits of the kappa B-binding protein NF-kappa B complex revealed a striking sequence homology of these proteins with the c-rel proto-oncogene product (c-Rel). On the basis of this homology, we examined the potential role of c-Rel in controlling IL-2R alpha transcription. We now demonstrate that the recombinant human c-Rel protein binds to the kappa B element in the IL-2R alpha promoter and results in alteration of the DNA structure in the adjacent downstream regulatory elements containing the CArG box and the GC box. We found that human c-Rel can activate transcription from the IL-2R alpha promoter, but not the kappa B-containing human immunodeficiency virus type 1 promoter, upon cotransfection into Jurkat T cells. Furthermore, truncation of the carboxyl terminus of c-Rel results in a c-Rel mutant (RelNA) that (i) localizes exclusively in the nucleus and (ii) acts in synergy with wild-type c-Rel in activating transcription from the kappa B site of the IL-2R alpha promoter. Finally, induction of surface IL-2R alpha expression coincides with the induced levels of endogenous c-Rel and induced c-Rel binding to the IL-2R alpha kappa B site. Our study identified c-Rel as one component of the Rel/NF-kappa B-family proteins involved in the kappa B-dependent activation of IL-2R alpha gene expression. Furthermore, our results suggest that a Re1NA-like cellular factor (e.g., NF-kappa B p50 or p49 subunit) acts in synergy with c-Re1 during T-cell activation.

Role of mitofusin 2 (Mfn2) in controlling cellular proliferation

It has been reported that Mitofusin2 (Mfn2) inhibits cell proliferation when overexpressed. We wanted to study the role of endogenous Mfn2 in cell proliferation, along with the structural features of Mfn2 that influence its mitochondrial localization and control of cell proliferation. Mfn2‐knockdown clones of a B‐cell lymphoma cell line BJAB exhibited an increased rate of cell proliferation. A 2‐fold increase in cell proliferation was also observed in Mfn2‐knockout mouse embryonic fibroblast (MEF) cells as compared with the control wild‐type cells, and the proliferative advantage of the knockout MEF cells was blocked on reintroduction of the Mfn2 gene. Mfn2 exerts its antiproliferative effect by acting as an effector molecule of Ras, resulting in the inhibition of the Ras‐Raf‐ERK signaling pathway. Furthermore, both the N‐terminal (aa 1–264) and the C‐terminal (aa 265–757) fragments of Mfn2 blocked cell proliferation through distinct mechanisms: the N‐terminal‐mediated inhibition was due to its interaction with Raf‐1, whereas the C‐terminal fragment of Mfn2 inhibited cell proliferation by interacting with Ras. The inhibition of proliferation by the N‐terminal fragment was independent of its mitochondrial localization. Collectively, our data provide new insights regarding the role of Mfn2 in controlling cellular proliferation.—Chen, K.‐H., Dasgupta, A., Ding, J., Indig, F. E., Ghosh, P., Longo, D. L. Role of Mitofusin 2 (Mfn2) in controlling cellular proliferation. FASEB J. 28, 382–394 (2014). www.fasebj.org

Comparison of the Effect of Mutant and Wild-Type p53 on Global Gene Expression

The mechanisms for “gain-of-function” phenotypes produced by mutant p53s such as enhanced proliferation, resistance to transforming growth factor-β–mediated growth suppression, and increased tumorigenesis are not known. One theory is that these phenotypes are caused by novel transcriptional regulatory events acquired by mutant p53s. Another explanation is that these effects are a result of an imbalance of functions caused by the retention of some of the wild-type transcriptional regulatory events in the context of a loss of other counterbalancing activities. An analysis of the ability of DNA-binding domain mutants A138P and R175H, and wild-type p53 to regulate the expression levels of 6.9 × 103 genes revealed that the mutants retained only <5% of the regulatory activities of the wild-type protein. A138P p53 exhibited mostly retained wild-type regulatory activities and few acquired novel events. However, R175H p53 possessed an approximately equal number of wild-type regulatory events and novel activities. This is the first report that, after examination of the regulation of a large unfocused set of genes, provides data indicating that remaining wild-type transcriptional regulatory functions existing in the absence of counterbalancing activities as well as acquired novel events both contribute to the gain-of-function phenotypes produced by mutant p53s. However, mutant p53s are likely to be distinct in terms of the extent to which each mechanism contributes to their gain-of-function phenotypes.

Interleukin-12-induced Interferon-γ Production by Human Peripheral Blood T Cells Is Regulated by Mammalian Target of Rapamycin (mTOR)

Depending on the type of external signals, T cells can initiate multiple intracellular signaling pathways that can be broadly classified into two groups based on their sensitivity to the immunosuppressive drug cyclosporin A (CsA). Interleukin (IL)-12-mediated interferon (IFN)-γ production by activated T cells has been shown to be CsA-insensitive. In this report, we demonstrate that the IL-12-induced CsA-resistant pathway of IFN-γ production is sensitive to rapamycin. Rapamycin treatment resulted in the aberrant recruitment of Stat3, Stat4, and phospho-c-Jun to the genomic promoter region resulting in decreased IFN-γ transcription. IL-12-induced phosphorylation of Stat3 on Ser-727 was affected by rapamycin, which may be due to the effect of rapamycin on the IL-12-induced interaction between mammalian target of rapamycin (mTOR) and Stat3. In accordance with this, reduction in the mTOR protein level by small interfering RNA resulted in suppression of Stat3 phosphorylation and decreased production of IFN-γ after IL-12 stimulation. These results suggest that mTOR may play a major role in IL-12-induced IFN-γ production by activated T cells.

Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of amyloid beta1−42 on neural progenitor cell proliferation

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ.

MOLECULAR REGULATION OF CYTOKINE GENE EXPRESSION : INTERFERON-GAMMA AS A MODEL SYSTEM

The regulation of IFN-gamma transcription appears to be quite complex. In addition to the interaction of numerous regions of the genomic DNA with multiple DNA binding protein family members, DNA methylation may serve to act as an early determinant of the capacity of a cell to initiate transcription. Transcriptional activation occurs in response to both soluble extracellular signals and cell contact, and it appears quite likely that this activation may result from the interaction of different families of DNA binding proteins with different enhancer elements. Furthermore, because chronic IFN-gamma transcription and subsequent expression would likely be detrimental to the host (see 81), mechanisms have evolved to quench expression at both transcriptional and posttranscriptional levels. Given the complexity of cell-to cell interactions in the immune system, it is reasonable to expect that additional mechanisms regulating IFN-gamma transcription, involving previously identified or as yet unidentified DNA binding proteins, remain to be defined.

Altered responsiveness of adenosine 3′,5′-monophosphate-generating systems in rat cortical slices after lesions of the medial forebrain bundle

The magnitude of the accumulation of radioactive cyclic AMP elicited by norepinephrine in [14C]adenine-labeled slices of rat cortical slices was enhanced 1.4-fold in ipsilateral slices from animals with unilateral lesions of the medial forebrain bundle as compared to slices from the contralateral hemisphere. This enhanced responsiveness to norepinephrine was first manifested 2 days after the lesion, and persisted for at least 3 weeks. The level of norepinephrine in cortical tissue from the hemisphere with lesion underwent a concomitant decline to less than 20% of the level in tissue from the contralateral hemisphere. Responses of cyclic AMP-generating systems to isoproterenol and to a combination of histamine and a phosphodiesterase inhibitor were also significantly enhanced in ipsilateral cortical slices after lesions of the medial forebrain bundle, while responses to a combination of norepinephrine and the β-adrenergic antagonist, propranolol, to adenosine, veratridine, prostaglandin E1, and a combination of serotonin and a phosphodiesterase inhibitor were not significantly altered. The response to histamine, in the presence of a phosphodiesterase inhibitor (isobutylmethylxanthine), was blocked effectively only by an H2-antagonist, metiamide. Responses to norepinephrine after lesions of the medial forebrain bundle were enhanced in ipsilateral hippocampal slices, but not in ipsilateral slices from midbrain or cerebellum.

Divergent effects of dithiocarbamates on AP-1-containing and AP-1-less NFAT sites.

Changes in the redox status of cells affect NF‐κB and activator protein (AP)‐1 nuclear expression and activity. In particular, antioxidants decrease NF‐κB and increase AP‐1 transcriptional activity, thereby regulating gene expression. In T cells, low concentrations of antioxidants enhance IL‐2 and inhibit IL‐4 expression. Since NFAT binding sites play an essential role in regulating IL‐2 and IL‐4 gene transcription, we studied the effects of dithiocarbamates, using the pyrrolidine derivative of dithiocarbamate (PDTC), on the activity of the distinct AP‐1‐containing IL‐2 NFAT and AP‐1‐less IL‐4 NFAT enhancers elements. Consistent with the presence of AP‐1 proteins within the IL‐2 NFAT complex, PDTC strongly enhanced phorbol 12‐myristate 13‐acetate/phytohemagglutinin‐induced NFAT binding to the IL‐2 NFAT enhancer and transcriptional activity of a reporter plasmid driven by this NFAT enhancer. In contrast, the activity of the IL‐4 NFp enhancer, which does not bind AP‐1, was abolished by PDTC treatment. In the Jurkat T cell line treated with PDTC, co‐expression of the Ca2+/calmodulin‐dependent phosphatase, calcineurin, completely restored the IL‐4 NFp enhancer activity. Our data indicate that calcineurin‐mediated NFAT activity is a target for antioxidants and provides new insights into the molecular mechanisms controlling differential cytokine gene expression.