Paritosh Ghosh

Mitofusin 2 (Mfn2), a mitochondrial protein, was shown to have antiproliferative properties when overexpressed. In this article, we show that activation of resting human peripheral blood T cells caused downregulation of Mfn2 levels. This downregulation of Mfn2 was blocked by different inhibitors (mTOR inhibitor rapamycin, PI3K inhibitor LY294002, and Akt inhibitor A443654), producing cells that were arrested in the G₀/G₁ stage of the cell cycle. Furthermore, the activation-induced downregulation of Mfn2 preceded the entry of the cells into the cell cycle, suggesting that Mfn2 downregulation is a prerequisite for activated T cell entry into the cell cycle. Accordingly, small interfering RNA–mediated knockdown of Mfn2 resulted in increased T cell proliferation. Overexpression of constitutively active AKT resulted in the downregulation of Mfn2, which can be blocked by a proteasome inhibitor. Akt-mediated downregulation of Mfn2 was via the mTORC1 pathway because this downregulation was blocked by rapamycin, and overexpression of wild-type, but not kinase-dead mTOR, caused Mfn2 downregulation. Our data suggested that activation-induced reactive oxygen species production plays an important role in the downregulation of Mfn2. Collectively, our data suggest that the PI3K-AKT-mTOR pathway plays an important role in activation-induced downregulation of Mfn2 and subsequent proliferation of resting human T cells.

The TORC1 pathway is necessary for ribosomal biogenesis and initiation of protein translation. Furthermore, the differentiation of Th1 and Th17 cells requires TORC1 activity. To investigate the role of the TORC1 pathway in the differentiation of Th1 and/or Th17 cells in more detail, we compared the differentiation capacity of naïve T cells from wild type and p70^S6K1 knockout mice. Expression of many of the genes associated with Th17‐cell differentiation, such as IL17a, IL17f, and IL‐23R, were reduced in p70^S6K1 knockout mice. In contrast, the development of Th1, Th2, and Treg cells was unaffected in the absence of p70^S6K1. Furthermore, expression of the major transcription factor in Th17‐cell differentiation, retinoic acid receptor‐related orphan receptor gamma T, remained unchanged. However, the acetylation of histone 3 at the promoters of IL17a and IL17f was reduced in the absence of p70^S6K1. In accordance with the in vitro data, the kinetics, but not the development, of EAE was affected with the loss of p70^S6K1 expression. Collectively, our findings suggested that both in vitro and in vivo differentiation of Th17 cells were positively regulated by p70^S6K1.

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

CXCL12 is a pleiotropic chemokine involved in multiple different processes such as immune regulation, inflammatory responses, and cancer development. CXCL12 is also a potent chemokine involved in chemoattraction of T cells to the site of infection or inflammation. Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that modulates different cellular processes, such as metabolism, nutrient sensing, protein translation, and cell growth. The role of mTOR in CXCL12-mediated resting T cell migration has yet to be elucidated. Rapamycin, an inhibitor of mTOR, significantly inhibits CXCL12 mediated migration of both primary human resting T cells and human T cell leukemia cell line CEM. p70^S6K1, an effector molecule of mTOR signaling pathway, was knocked down by shRNA in CEM cells using a lentiviral gene transfer system. Using p70^S6K1 knock down cells, we demonstrate the role of mTOR signaling in T cell migration both in vitro and in vivo. Our data demonstrate a new role for mTOR in CXCL12-induced T cell migration, and enrich the current knowledge regarding the clinical use of rapamycin.

The role of T cells in innate immunity is not well defined. In this report, we show that a subset of human peripheral blood CD4⁺ T cells responds to IL-12 plus IL-18, but not to IL-12 or IL-18 alone, by producing IFN-γ in the absence of any antigenic stimulation or cell proliferation. Intracellular staining reveals a small percentage of resting CD4⁺ T cells (0.5 to 1.5%) capable of producing IFN-γ in response to IL-12 plus IL-18. Interestingly, both naïve (CD45RA⁺) and memory (CD45RO⁺) CD4⁺ populations were responsive to IL-12 plus IL-18 stimulation in producing IFN-γ. The expression of IFN-γinduced by IL-12 and IL-18 is sensitive to rapamycin and SB203580, indicating the possible involvement of mTOR and p38 MAP kinase, respectively, in this synergistic pathway. While p38MAP kinase is involved in transcription, mTOR is involved in message stabilization. We have also shown that NFκB family member, cRel, but not GADD45β and GADD45γ, plays an important role in IL-12 plus IL-18-induced IFN-γ transcription. Thus, the present study suggests that naïve CD4⁺ T cells may participate in innate immunity or amplify adaptive immune responses through cytokine-induced antigen-independent cytokine production.

There are multiple mechanisms by which cells evade TGF-β–mediated growth inhibitory effects. In this report, we describe a novel mechanism by which cells become resistant to TGF-β–mediated growth suppression. Although having all the components of the TGF-β signaling pathway, different cell lines, RL, HaCaT, and BJAB, have different sensitivities toward TGF-β–induced growth suppression. The TGF-β resistance of RL, a B-cell lymphoma cell line, was due to ligand-induced downregulation of TGF-β receptor II (TβRII) and only transient TGF-β induced nuclear translocation of Smad2 and Smad3. With low-dose phorbol 12-myristate 13-acetate (PMA) or anti-IgM treatment, TGF-β sensitivity was restored by stabilizing TβRII expression and sustaining TGF-β signaling. The MEK inhibitor, U0126, blocked both PMA- and anti-IgM–induced upregulation of TβRII. In HaCaT and BJAB, two TGF-β–sensitive cell lines, which had higher basal levels of phospho-MEK and TβRII compared with RL, U0126 induced downregulation of TβRII and blocked subsequent TGF-β signaling. Similar results were also obtained with normal B cells, where MEK1 inhibitor downregulated TβRII and subsequent TGF-β signaling. Constitutively active MEK1, but not constitutively active ERK2, induced upregulation of TβRII. Furthermore, TβRII physically interacted with the constitutively active MEK1, but not with wild-type MEK1, indicating involvement of active MEK1 in stabilizing TβRII. Collectively, our data suggest a novel mechanism for MEK1 in regulating the sensitivity to TGF-β signaling by stabilizing TβRII. Mol Cancer Res; 9(1); 78–89 ©2010 AACR.

The consequences of T-cell activation depend exclusively on costimulation during antigen–T-cell receptor interaction. Interaction between the T-cell coreceptor CD28 and its ligand B7 during antigen-antigen receptor engagement results in full activation of T cells, the outcomes of which are proliferation and effector functions. The ability of CD28 to costimulate the production of interleukin-2 (IL-2) explains the importance of this costimulation. The signaling event mediated by CD28 engagement has been proposed to have 2 components: one is sensitive to the immunosuppressive drug cyclosporin A (CsA), and the other one is CsA-resistant. In this report, we demonstrate that the CsA-resistant pathway is sensitive to the immunosuppressive drug rapamycin. Treatment with rapamycin blocked IL-2 production after activation of human peripheral blood T cells with phorbol ester (PMA) and anti-CD28 (CsA-resistant pathway), whereas this drug did not have any effect on PMA plus ionomycin stimulation (CsA-sensitive pathway). The inhibitory effect of rapamycin was on messenger RNA stability and translation, rather than on IL-2 transcription or protein turnover.

Background:The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by T helper 1 (Th1) and T helper 2(Th2) T-cell subsets, which are two arms of the immune system. Th1 cells preferentially produce interleukin 2(IL-2), interferon gamma (IFN γ), and tumor necrosis factor (TNF), 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 and stimulate an antibody response that helps to prevent the cells from becoming infected. The clinical progression of several infectious diseases, including human immuno deficiency virus, some types of parasitoses, and tuberculosis, is thought to be associated with the predominance of a Th2-type T-cell response. Recent reports have demonstrated the presence of T cells producing Th2 lymphokines(IL-4, IL-6, and IL-10) in tumor-infiltrating lymphocytes of renal cell carcinoma.Purpose:The purpose of thisstudy was to investigate at themolecular level whether there was anychange in the splenic T cells of mice with progressively growing tumors from a Thl to a Th2 DNA-binding patternor phenotype.Methods:Splenic Tcells from mice bearing renal cell carcinomaor MCA-38 colon carcinoma were tested for cytokine production after in vitro activation. Nuclear extracts of splenic T cells were used for the DNA-binding assay using IFN-γ core promoter region, the kappa B(kB) site from immunoglobulin gene, and the nuclear factor of activated Tcell (NFAT) site from IL-2 gene.Results:Splenic T cells from mice bearing renal cell carcinoma or MCA-38 colon carcinoma preferentially produced Th2 cytokines (i.e., IL-4) upon activation and showed a marked decrease in Th1 cytokine (particularly IFN y) production compared with the production observed in normal splenic T cells. The DNA-binding assay with the IFN-γ core promoter region confirmed the gradual decline in the nuclear transcription factors associated with the Th1 pheno type during tumor progression in both tumor models. Renal cell carcinoma-bearing mice,successfully treated with flavone-8-acetic acid and recombinant human IL-2, showed a reversion to a Th1-likepattern. In addition, nuclear extracts of T cells from tumor-bearing animals showed a Th2-type KB-binding pattern. Moreover, the NFAT complex present in the normal splenic T cells was lost at the later stages of tumor progression; instead, a new complex was present in mice bearing long-term tumors.Conclusion:T cells from tumor-bearing mice lose the Th1 phenotype with progressive tumor growth. [J Natl Cancer Inst 1995;87:1478–83]