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In JoVE (1)
Other Publications (15)
- Journal of the National Cancer Institute
- Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research
- Cancer Cell
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Cellular Signalling
- Immunity
- The Journal of Biological Chemistry
- Molecular Biology of the Cell
- Cancer Biology & Therapy
- Molecular Cancer Research : MCR
- Cancer Prevention Research (Philadelphia, Pa.)
- Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research
- The Journal of Biological Chemistry
- Journal of Interferon & Cytokine Research : the Official Journal of the International Society for Interferon and Cytokine Research
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Articles by Ana M. Gamero in JoVE
JoVE Clinical and Translational Medicine
Cecal Ligation Punktering Förfarande
1Department of Microbiology and Immunology School of Medicine, Temple University, 2Department of Biochemistry, School of Medicine, Temple University
Den musmodell av cecal ligation och punktera som ett värdefullt verktyg för studiet av mänskliga sepsis.
Other articles by Ana M. Gamero on PubMed
Effects of Interferon Beta on Transcobalamin II-receptor Expression and Antitumor Activity of Nitrosylcobalamin
The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl.
Thrombomodulin RNA is Destabilized Through Its 3'-untranslated Element in Cells Exposed to IFN-gamma
Interferon-gamma (IFN-gamma) is a potent activator of mononuclear phagocytes, allowing them to play a prominent role in acute and chronic inflammatory responses. IFN-gamma binding to its cell surface receptor initiates changes in the steady-state levels of cellular RNAs, permitting the proteins encoded by these RNAs to exert its biologic actions. Hundreds of cellular RNAs have been identified whose rates of transcription are altered by incubation of cells with IFNs. The rates of transcription of many of the genes encoding these RNAs are enhanced by IFN-gamma-mediated activation of the Stat1 transcription factor that is tyrosine phosphorylated and translocates to the nucleus, where it binds enhancers present in IFN-stimulated genes (ISGs). IFN-gamma can also modify the concentrations of some RNAs by posttranscriptional mechanisms. However, very little is understood about the molecular mechanisms regulating this phenomenon. We have identified the RNA encoding thrombomodulin (TM), a physiologic receptor for thrombin, that is downregulated in primary human monocytes incubated with IFN-gamma. Using actinomycin D as a transcriptional inhibitor, we show that the mRNA half-life is rapidly shortened by IFN-gamma. The TM transcript contains a large 3'-untranslated region (UTR), with several AU-rich elements (AREs), elements that have been implicated in the regulation of mRNA decay. Using a tetracycline-regulatory promoter system, we analyzed RNA levels in the absence of transcription of TM. Results from these experiments indicate that incubation of cells with IFN-gamma accelerates the decay of TM RNA through its 3'-UTR. This is the first report describing a clear posttranscriptional downregulation of an mRNA by IFN-gamma that identifies the 3'-UTR as a target of IFN-gamma-stimulated destabilization.
Inactivation of Stat3 in Tumor Cells: Releasing a Brake on Immune Responses Against Cancer?
A model of immune evasion mediated by tumors expressing constitutively activated Stat3 was recently proposed in Nature Medicine by Wang et al., suggesting opportunities for a new therapeutic approach for cancer.
Identification of a Novel Conserved Motif in the STAT Family That is Required for Tyrosine Phosphorylation
The rapid transcriptional activation of cellular genes by either type 1 interferons (IFNalpha/beta) or type 2 interferon (IFNgamma) is responsible for many of the pleiotropic effects of these cytokines, including their antiviral, antigrowth, and immunomodulatory activities. Interferon-stimulated gene expression is mediated by transcription factors termed Stats, which upon being tyrosine-phosphorylated, translocate to the nucleus and bind enhancers of interferon-activated genes. We have recently characterized a new Jurkat cell variant, named H123, where IFNalpha stimulates programmed cell death. H123 clones that are resistant to the apoptotic actions of IFNalpha have been selected. One of these clones (Clone 8) is defective in its responses to IFNalpha with regard to activation of genes that require tyrosine phosphorylation of Stat2. Stimulation of Clone 8 cells with IFNalpha induces normal tyrosine phosphorylation of Stat1 and Stat3. Sequencing of Stat2 RNA reveals a substitution of proline 630 located within the Src homology 2 domain of Stat2 to leucine (P630L). Pro-630 and its adjacent amino acids are conserved in all Stat family members but are absent in other proteins that contain Src homology 2 domains. Expression of Stat2 P630L in cells inhibits IFNalpha-stimulated gene expression. These results not only define a critical motif in Stat2 required for its transcriptional activity, but they also provide evidence that resistance to type one IFNs can be mediated by mutations in Stat2 as well as those previously described for Stat1.
Calcium-dependent Activation of Interleukin-21 Gene Expression in T Cells
Interleukin (IL)-21 is a gamma(c)-dependent cytokine produced by activated T cells with important actions for T, B, and NK cells. The IL-21 gene is adjacent to the IL-2 gene, and like IL-2, IL-21 is strongly induced at the transcriptional level after T cell activation. Interestingly, however, in contrast to the IL-2 gene, a calcium ionophore alone was sufficient to induce IL-21 gene expression in preactivated T cells. Two DNase I hypersensitivity sites were found in the IL-21 gene, corresponding to nucleotide sequences that are conserved in humans and mice. One site is located at the IL-21 promoter region and conferred T cell receptor-mediated IL-21 gene transcription. TCR-induced IL-21 gene expression was inhibited by cyclosporin A and FK506. Correspondingly, the IL-21 5'-regulatory region contains three NFAT binding sites, and induction of IL-21 promoter activity was impaired when these sites were mutated or following treatment with cyclosporin A. Thus, our studies reveal that in contrast to IL-2, a calcium signal alone is sufficient to mediate induction of the IL-21 in preactivated T lymphocytes and that this induction appears to result from specific NFAT binding.
Type I Interferons Activate Apoptosis in a Jurkat Cell Variant by Caspase-dependent and Independent Mechanisms
Although the antiviral actions of interferons (IFNs) are observed in most types of cells, the antiproliferative effects of IFNalpha/beta are variable as are the mechanisms of growth inhibition that may or may not be due to the induction of apoptosis. To understand more about the mechanisms that are responsible for IFNalpha/beta-stimulated apoptosis, we have characterized a new human Jurkat T cell variant named H123 where IFNalpha activates programmed cell death (PCD). No differences in IFNalpha-stimulated, Stat-dependent gene expression were detected between H123 cells and the parental Jurkat cells, which are growth inhibited, but do not undergo apoptosis with IFNalpha. Although IFNalpha stimulates the activity of both caspase 3 and 9 in H123 cells, the general caspase inhibitor Z-VAD only partially reverses the apoptotic actions of IFNalpha. Induction of apoptosis by IFNalpha occurs through a mitochondrial-dependent pathway in H123 cells, as demonstrated by the release of cytochrome C from the mitochondria. Furthermore, IFNalpha treatment of H123 cells stimulates the release of the serine protease HtrA2/Omi from the mitochondria, suggesting that it plays a role in the apoptotic actions of this cytokine. These results provide evidence for a novel type 1 IFN-mediated pathway that regulates apoptosis of T cells through a mitochondrial-dependent and caspase-dependent and independent pathway.
IL-1 Can Act As Number One
Both interleukin-1 receptor (IL-1R) and toll-like receptor 2 (TLR2) signal via the adapter molecule MyD88. However, in response to a cutaneous infection challenge, the downstream signaling events diverge and IL-1R, but not TLR2, proves to be essential for host defense.
Activation of Tyk2 and Stat3 is Required for the Apoptotic Actions of Interferon-beta in Primary Pro-B Cells
The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.
A Mutation in the SH2 Domain of STAT2 Prolongs Tyrosine Phosphorylation of STAT1 and Promotes Type I IFN-induced Apoptosis
Type I interferons (IFN-alpha/beta) induce apoptosis in certain tumor cell lines but not others. Here we describe a mutation in STAT2 that confers an apoptotic effect in tumor cells in response to type I IFNs. This mutation was introduced in a conserved motif, PYTK, located in the STAT SH2 domain, which is shared by STAT1, STAT2, and STAT3. To test whether the tyrosine in this motif might be phosphorylated and affect signaling, Y631 of STAT2 was mutated to phenylalanine (Y631F). Although it was determined that Y631 was not phosphorylated, the Y631F mutation conferred sustained signaling and induction of IFN-stimulated genes. This prolonged IFN response was associated with sustained tyrosine phosphorylation of STAT1 and STAT2 and their mutual association as heterodimers, which resulted from resistance to dephosphorylation by the nuclear tyrosine phosphatase TcPTP. Finally, cells bearing the Y631F mutation in STAT2 underwent apoptosis after IFN-alpha stimulation compared with wild-type STAT2. Therefore, this mutation reveals that a prolonged response to IFN-alpha could account for one difference between tumor cell lines that undergo IFN-alpha-induced apoptosis compared with those that display an antiproliferative response but do not die.
IFNalpha and IFNlambda Differ in Their Antiproliferative Effects and Duration of JAK/STAT Signaling Activity
Interferon (IFN)lambda, also known as IL-28A, IL-28B or IL-29, is a new type III IFN, which like type I IFN(alpha/beta), activates common elements of the JAK/STAT signaling pathway and exhibits antiproliferative activity. Currently, IFNalpha is used in the treatment of certain forms of cancer, but its antitumor effects are limited and associated with high toxicity. In this study, we determined whether IFNlambda induced the same level of cell growth inhibition relative to IFNalpha. To this effect HaCaT cells, which are typically growth inhibited by IFNalpha, underwent apoptosis in response to IFNlambda. Next, in contrast to IFNalpha stimulation, IFNlambda prolonged the duration of activated STAT1 and STAT2. Furthermore, the kinetics of IFN-stimulated genes was different as IFNlambda induced a delayed but stronger induction of IFN-responsive genes. Components of the JAK/STAT pathway remained essential for the antiproliferative effects of IFNalpha and IFNlambda. IFNlambda-induced persistence of STAT activation required de novo protein synthesis and was in part due to a delay in STAT2 inactivation. Thus our data demonstrate that the duration of IFNlambda signaling is different from that of IFNalpha, and that IFNlambda could be a suitable cytokine to evaluate for cancer therapy.
Resistance to IFN-alpha-induced Apoptosis is Linked to a Loss of STAT2
Type I IFNs (IFN-alpha/beta) are pleitropic cytokines widely used in the treatment of certain malignancies, hepatitis B and C, and multiple sclerosis. IFN resistance is a challenging clinical problem to overcome. Hence, understanding the molecular mechanism by which IFN immunotherapy ceases to be effective is of translational importance. In this study, we report that continuous IFN-alpha stimulation of the human Jurkat variant H123 led to resistance to type I IFN-induced apoptosis due to a loss of signal transducers and activators of transcription 2 (STAT2) expression. The apoptotic effects of IFN-alpha were hampered as STAT2-deficient cells were defective in activating the mitochondrial-dependent death pathway and ISGF3-mediated gene activation. Reconstitution of STAT2 restored the apoptotic effects of IFN-alpha as measured by the loss of mitochondrial membrane potential, cytochrome c release from mitochondria, caspase activation, and ultimately cell death. Nuclear localization of STAT2 was a critical event as retention of tyrosine-phosphorylated STAT2 in the cytosol was not sufficient to activate apoptosis. Furthermore, silencing STAT2 gene expression in Saos2 and A375S.2 tumor cell lines significantly reduced the apoptotic capacity of IFN-alpha. Altogether, we show that STAT2 is a critical mediator in the activation of type I IFN-induced apoptosis. More importantly, defects in the expression or nuclear localization of STAT2 could lessen the efficacy of type I IFN immunotherapy.
STAT2 Contributes to Promotion of Colorectal and Skin Carcinogenesis
Signal transducer and activator of transcription 2 (STAT2) is an essential transcription factor in the type I IFN (IFN-alpha/beta) signal transduction pathway and known for its role in mediating antiviral immunity and cell growth inhibition. Unlike other members of the STAT family, IFNs are the only cytokines known to date that can activate STAT2. Given the inflammatory and antiproliferative dual nature of IFNs, we hypothesized that STAT2 prevents inflammation-induced colorectal and skin carcinogenesis by altering the inflammatory immune response. Contrary to our hypothesis, deletion of STAT2 inhibited azoxymethane/dextran sodium sulfate-induced colorectal carcinogenesis as measured by prolonged survival, lower adenoma incidence, smaller polyps, and less chronic inflammation. STAT2 deficiency also inhibited 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis as indicated by reduced papilloma multiplicity. A potential mechanism by which STAT2 promotes carcinogenesis is through activation of proinflammatory mediators. Deletion of STAT2 decreased azoxymethane/dextran sodium sulfate-induced expression and release of proinflammatory mediators, such as interleukin-6 and CCL2, and decreased interleukin-6 release from skin carcinoma cells, which then decreased STAT3 activation. Our findings identify STAT2 as a novel contributor to colorectal and skin carcinogenesis that may act to increase the gene expression and secretion of proinflammatory mediators, which in turn activate the oncogenic STAT3 signaling pathway.
Interferon-lambda As a Potential Therapeutic Agent in Cancer Treatment
The discovery that type I interferon (IFN-alpha/beta) inhibited tumor cell growth was welcomed initially with great excitement as it rapidly became a U.S. Food and Drug Administration-approved drug to treat several forms of cancer. In time, this enthusiasm diminished as severe toxicity associated with IFN-alpha administration, resistance to the therapy, or less than optimal responses became evident in cancer patients, thus restricting its clinical use and reducing its potential as an anticancer drug. The recent discovery of a third type of IFN [IFN-lambda/interleukin (IL)-29/IL-28], which shares the same biological properties of type I IFNs, opens the door for evaluating the therapeutic potential of IFN-lambda as it uses a distinct receptor complex whose expression, unlike type I IFN receptors, is restricted to cells of specific lineage. It is unclear whether the mechanism by which type III IFNs restrict tumor cell proliferation is different or the same from the one utilized by type I IFN. Nevertheless, accumulating evidence as described in this review suggests that, in contrast to IFN-alpha therapy, IFN-lambda therapy could be less toxic and suitable for certain types of malignancies as not all cells are responsive to this cytokine.
Control of Type I Interferon-induced Cell Death by Orai1-mediated Calcium Entry in T Cells
Store-operated Ca(2+) entry (SOCE) is an essential process in T cell activation. SOCE is controlled by the Ca(2+) release-activated Ca(2+) (CRAC) channel encoded by the gene Orai1 that is expressed on the plasma membrane and activated by STIM1 when ER Ca(2+) stores are depleted. Our earlier work showed that a somatic T-cell line Jurkat mutant H123 bearing a defect in Ca(2+) signaling was susceptible to the apoptotic effects of type I interferons (IFN-α/β). The nature of the mutation and whether this mutation was linked to IFN-α/β apoptotic susceptibility was unknown. Here we show that H123 cells lacked Orai1 and exhibit reduced STIM1 protein. Reconstitution of both Orai1 and STIM1 in H123 cells rescued SOCE in response to thapsigargin and ionomycin and abrogated IFN-α/β-induced apoptosis. Reciprocally, overexpression of the dominant negative Orai1-E106A in either parental Jurkat cells or an unrelated human T cell line (CEM391) inhibited SOCE and led to sensitization to IFN-α/β-induced apoptosis. Furthermore, we showed that the Ca(2+) response pathway antagonized the IFN-α/β -induced transcriptional responses; in the absence of SOCE, this negative regulatory effect was lost. However, the inhibitory effect of Ca(2+) on type I IFN-induced gene transcription was diminished by pharmacological inhibition of NF-κB in cells with intact SOCE. Our findings reveal an unexpected and novel regulatory crosstalk mechanism between type I IFNs and store-operated Ca(2+) signaling pathways mediated at least in part by NF-κB activity with significant clinical implications to both viral and tumor immunology.
The Role of Signal Transducer and Activator of Transcription-2 in the Interferon Response
The signal transducer and activator of transcription-2 (STAT2) was discovered as a cellular component of the DNA binding complex known as interferon (IFN) stimulated gene factor-3. Numerous studies have confirmed that STAT2 operates as a positive regulator in the transcriptional activation response elicited by IFNs. In this article, we highlight the progress made in elucidating the pivotal role of STAT2 in driving the expression of IFN-induced genes, innate antiviral immunity, apoptosis, and cancer. A better understanding of the functional role of STAT2 in the IFN response and how STAT2 is regulated will uncover new clues to its role in diseases.
