Biomedical subjects

Antonella Sassano

Publications and source records attributed to Antonella Sassano.

At least 19 recordsLinked to original sources

PubMed2025

Long-read sequencing of single cell-derived melanoma subclones reveals divergent and parallel genomic and epigenomic evolutionary trajectories.

Tumor evolution is driven by various mutational processes, ranging from single-nucleotide variants (SNVs) to large structural variants (SVs) to dynamic shifts in DNA methylation. Current short-read sequencing methods struggle to accurately capture the full spectrum of these genomic and epigenomic alterations due to inherent technical limitations. To overcome that, here we introduce an approach for long-read sequencing of single-cell derived subclones, and use it to profile 23 subclones of a mouse melanoma cell line, characterized with distinct growth phenotypes and treatment responses. We develop a computational framework for harmonization and joint analysis of different variant types in the evolutionary context. Uniquely, our framework enables detection of recurrent amplifications of putative driver genes, generated by independent SVs across different lineages, suggesting parallel evolution. In addition, our approach revealed gradual and lineage-specific methylation changes associated with aggressive clonal phenotypes. We also show our set of phylogeny-constrained variant calls along with openly released sequencing data can be a valuable resource for the development of new computational methods.

Journal Article↗

PubMed2024

Stochastic modeling of single-cell gene expression adaptation reveals non-genomic contribution to evolution of tumor subclones.

Cancer progression is an evolutionary process driven by the selection of cells adapted to gain growth advantage. We present a formal study on the adaptation of gene expression in subclonal evolution. We model evolutionary changes in gene expression as stochastic Ornstein-Uhlenbeck processes, jointly leveraging the evolutionary history of subclones and single-cell expression data. Applying our model to sublines derived from single cells of a mouse melanoma revealed that sublines with distinct phenotypes are underlined by different patterns of gene expression adaptation, indicating non-genetic mechanisms of cancer evolution. Sublines previously observed to be resistant to anti-CTLA4 treatment showed adaptive expression of genes related to invasion and non-canonical Wnt signaling, whereas sublines that responded to treatment showed adaptive expression of genes related to proliferation and canonical Wnt signaling. Our results suggest that clonal phenotypes emerge as the result of specific adaptivity patterns of gene expression. A record of this paper's transparent peer review process is included in the supplemental information.

Animals↗

PubMed2006

Regulatory effects of mammalian target of rapamycin-activated pathways in type I and II interferon signaling.

The mechanisms regulating initiation of mRNA translation for the generation of protein products that mediate interferon (IFN) responses are largely unknown. We have previously shown that both Type I and II IFNs engage the mammalian target of rapamycin (mTOR), resulting in downstream phosphorylation and deactivation of the translational repressor 4E-BP1 (eIF4E-binding protein 1). In the current study, we provide direct evidence that such regulation of 4E-BP1 by IFNalpha or IFNgamma results in sequential dissociation of 4E-BP1 from eukaryotic initiation factor-4E and subsequent formation of a functional complex between eukaryotic initiation factor-4E and eukaryotic initiation factor-4G, to allow initiation of mRNA translation. We also demonstrate that the induction of key IFNalpha- or IFNgamma-inducible proteins (ISG15 (interferon-stimulated gene 15) and CXCL10) that mediate IFN responses are enhanced in 4E-BP1 (4E-BP1(-/-)) knockout MEFs, as compared with wild-type 4E-BP1(+/+) MEFs. On the other hand, IFN-dependent transcriptional regulation of the Isg15 and Cxcl10 genes is intact in the absence of 4E-BP1, as determined by real time reverse transcriptase-PCR assays and promoter assays for ISRE and GAS, establishing that 4E-BP1 plays a selective negative regulatory role in IFN-induced mRNA translation. Interestingly, the induction of expression of ISG15 and CXCL10 proteins by IFNs was also strongly enhanced in cells lacking expression of the tuberin (TSC2(-/-)) or hamartin (TSC1(-/-)) genes, consistent with the known negative regulatory effect of the TSC1-TSC2 complex on mTOR activation. In other work, we demonstrate that the induction of an IFN-dependent antiviral response is strongly enhanced in cells lacking expression of 4E-BP1 and TSC2, demonstrating that these elements of the IFN-activated mTOR pathway exhibit important regulatory effects in the generation of IFN responses. Taken altogether, our data suggest an important role for mTOR-dependent pathways in IFN signaling and identify 4E-BP1 and TSC1-TSC2 as key components in the generation of IFN-dependent biological responses.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Role of the p38 mitogen-activated protein kinase pathway in the generation of arsenic trioxide-dependent cellular responses.

Arsenic trioxide (As(2)O(3)) induces differentiation and apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms that mediate such effects are not known. In the present study, we provide evidence that the kinases MAPK kinase 3 (Mkk3) and Mkk6 are activated during treatment of leukemic cell lines with As(2)O(3) to regulate downstream engagement of the p38 mitogen-activated protein kinase. Using cells with targeted disruption of both the Mkk3 and Mkk6 genes, we show that As(2)O(3)-dependent activation of p38 is defective in the absence of Mkk3 and Mkk6, establishing that these kinases are essential for As(2)O(3)-dependent engagement of the p38 pathway. Pharmacologic inhibition of p38 enhances As(2)O(3)-dependent activation of the c-jun NH(2)-terminal kinase (JNK) and subsequent induction of apoptosis of chronic myelogenous leukemia (CML)- or acute promyelocytic leukemia (APL)-derived cell lines. In addition, in APL blasts, inhibition of p38 enhances myeloid cell differentiation in response to As(2)O(3), as well as suppression of Bcl-2 expression and loss of mitochondrial membrane potential. Similarly, induction of As(2)O(3)-dependent apoptosis is enhanced in mouse embryonic fibroblasts (MEF) with targeted disruption of both the Mkk3 and Mkk6 genes, establishing a key role for this pathway in the regulation of As(2)O(3)-induced apoptosis. In other studies, we show that the small-molecule p38 inhibitors SD-282 and SCIO-469 potentiate As(2)O(3)-mediated suppression of myeloid leukemic progenitor growth from CML patients, indicating a critical regulatory role for p38 in the induction of antileukemic responses. Altogether, our data indicate that the Mkk3/6-p38 signaling cascade is activated in a negative regulatory feedback manner to control induction of As(2)O(3)-mediated antileukemic effects.

Animals↗

PubMed2006

Recombinant human uteroglobin/CC10 inhibits the adhesion and migration of primary human endothelial cells via specific and saturable binding to fibronectin.

Uteroglobin (UG) or Clara Cell 10 kDa protein (CC10) is a small, stable, epithelial secretory anti-inflammatory protein. Uteroglobin has been shown to inhibit neointimal formation in vivo after balloon angioplasty through an unknown mechanism. An interaction between UG and plasma fibronectin (Fn) has been demonstrated in mice. Since Fn plays a key role in endothelial cell (EC) migration and angiogenesis, we investigated whether recombinant human UG (rhUG) affects EC migration via Fn binding. In this report, we show a saturable binding of rhUG to Fn depending on Fn conformation and that rhUG is covalently cross-linked to Fn by transglutaminase (TGase). Additionally, our study highlights that rhUG can also bind to exogenously added or self-secreted Fn on the membrane of human primary microvascular endothelial cells (HMVEC), although these complexes are weakly associated with the plasmalemma. Upon the interaction with Fn in solid phase, rhUG strongly inhibits HMVEC attachment on Fn, but not on other ECM proteins. Consequently, rhUG also inhibits cell migration in a dose dependent fashion (I.C.50 = 65 nM) and hinders the "wound healing" in vitro. The small size, stability and human tolerability of rhUG suggest that rhUG in slow-release form or genetically delivered could be used in humans to modulate cell/Fn interactions in the context of tumor microenvironment or in the context of inflammation and fibrosis.

Binding, Competitive↗

PubMed2006

Vaccinia virus activation of CCR5 invokes tyrosine phosphorylation signaling events that support virus replication.

Vaccinia virus, a poxvirus, produces structurally distinct forms of virions for which the immediate events following cell entry are ill-defined. We provide evidence that intracellular mature virus (IMV) enters both permissive and nonpermissive T-cell lines and that introduction of CCR5 into nonpermissive mouse fibroblasts or human primary T cells renders the cells permissive for vaccinia replication. Notably, T cells expressing CCR5 in which tyrosine 339 in the intracellular region is replaced by phenylalanine no longer support virus replication or virus-inducible activation of specific host cell signaling effectors IRS-2, Grb2, and Erk1/2. We show that following IMV entry into the cell, the intact but not the tyrosine-deficient CCR5 is rapidly internalized and colocalizes with virus. This colocalization precedes virus-inducible signaling and replication.

Amino Acid Substitution↗

PubMed2006

Activation of mammalian target of rapamycin and the p70 S6 kinase by arsenic trioxide in BCR-ABL-expressing cells.

Arsenic trioxide (As(2)O(3)) exhibits important antitumor activities in vitro and in vivo, but the precise mechanisms by which it induces its effects are not known. We provide evidence that during treatment of BCR-ABL-expressing cells with As(2)O(3), there is activation of a cellular pathway involving the p70 S6 kinase (p70S6K). Our data show that p70S6K is rapidly phosphorylated on Thr(421) and Ser(424) and is activated in an As(2)O(3)-inducible manner. The mammalian target of rapamycin (mTOR) is also phosphorylated/activated in an As(2)O(3)-inducible manner, and its activity is required for downstream engagement of p70S6K. p70S6K subsequently phosphorylates the S6 ribosomal protein on Ser(235)/Ser(236) and Ser(240)/Ser(244) to promote initiation of mRNA translation. Treatment of chronic myelogenous leukemia-derived cell lines with As(2)O(3) also results in phosphorylation of the 4E-BP1 repressor of mRNA translation on Thr(37)/Thr(46) and Thr(70), sites required for its deactivation and its dissociation from the eukaryotic initiation factor 4E complex to allow cap-dependent mRNA translation. In studies to determine the functional relevance of this pathway, we found that inhibition of mTOR and downstream cascades enhances induction of apoptosis by As(2)O(3). Consistent with this, the mTOR inhibitor rapamycin strongly potentiated As(2)O(3)-mediated suppression of primitive leukemic progenitors from the bone marrow of chronic myelogenous leukemia patients. Altogether, our data show that the mTOR/p70S6K pathway is activated in a negative feedback regulatory manner in response to As(2)O(3) in BCR-ABL-transformed cells and plays a key regulatory role in the induction of anti-leukemic responses.

Antineoplastic Agents↗

PubMed2005

Role of the p38 mitogen-activated protein kinase pathway in cytokine-mediated hematopoietic suppression in myelodysplastic syndromes.

The p38 mitogen-activated protein kinase (MAPK) pathway is activated by IFNs and other cytokines to mediate signals for important cellular functions, including transcriptional regulation and apoptosis. We examined the role of the p38 pathway in the generation of the effects of myelosuppressive cytokines on human hematopoiesis. Pharmacologic inhibition of p38 using BIX-01208 resulted in reversal of IFN-, tumor necrosis factor-alpha (TNF-alpha)-, and transforming growth factor-beta (TGF-beta)-mediated suppression of human erythroid (blast-forming unit-erythroid) and myeloid (granulocyte-macrophage colony-forming unit) colony formation, consistent with a key role for p38 in the generation of myelosuppressive signals by different cytokines. Similarly, the myelosuppressive effects of TNF-alpha and TGF-beta were reversed by small interfering RNAs targeting p38alpha expression, further establishing the requirement of this kinase in the induction of myelosuppressive responses. As TNF overproduction has been implicated in the pathophysiology of bone marrow failure states, we determined whether pharmacologic inhibition of p38 reverses the hematopoietic defects seen in bone marrows from patients with myelodysplastic syndromes (MDS) and the anemia of chronic disease. Addition of pharmacologic inhibitors of p38 on such bone marrows resulted in increased numbers of erythroid and myeloid progenitors. Similarly, inhibition of the activity of the downstream effectors of p38, MAPK activated protein kinase-2, and mitogen and stress activated kinase 1 partially restored the hematopoietic defect seen in these bone marrows. Taken altogether, our data implicate the p38 MAPK in the pathophysiology of myelodysplasias and suggest that p38 pharmacologic inhibitors may have therapeutic applications in the treatment of MDS.

Aged↗

PubMed2005

Differential regulation of the p70 S6 kinase pathway by interferon alpha (IFNalpha) and imatinib mesylate (STI571) in chronic myelogenous leukemia cells.

The precise mechanisms by which imatinib mesylate (STI571) and interferon alpha (IFNalpha) exhibit antileukemic effects are not known. We examined the effects of IFNs or imatinib mesylate on signaling pathways regulating initiation of mRNA translation in BCR-ABL-expressing cells. Treatment of IFN-sensitive KT-1 cells with IFNalpha resulted in phosphorylation/activation of mammalian target of rapamycin (mTOR) and downstream activation of p70 S6 kinase. The IFN-activated p70 S6 kinase was found to regulate phosphorylation of S6 ribosomal protein, which regulates translation of mRNAs with oligopyrimidine tracts in the 5'-untranslated region. In addition, IFNalpha treatment resulted in an mTOR- and/or phosphatidyl-inositol 3'(PI 3') kinase-dependent phosphorylation of 4E-BP1 repressor of mRNA translation on sites that are required for its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. In contrast to the effects of IFNs, imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Moreover, the mTOR inhibitor rapamycin enhanced the suppressive effects of imatinib mesylate on primary leukemic granulocyte macrophage-colony-forming unit (CFU-GM) progenitors from patients with chronic myelogenous leukemia (CML). Taken altogether, our data demonstrate that IFNs and imatinib mesylate differentially regulate PI 3' kinase/mTOR-dependent signaling cascades in BCR-ABL-transformed cells, consistent with distinct effects of these agents on pathways regulating mRNA translation. They also support the concept that combined use of imatinib mesylate with mTOR inhibitors may be an appropriate future therapeutic strategy for the treatment of CML.

5' Untranslated Regions↗

PubMed2005

Activation of mitogen-activated protein kinase kinase (MKK) 3 and MKK6 by type I interferons.

There is accumulating evidence that the p38 MAP kinase pathway plays important roles in Type I interferon (IFN) signaling, but the mechanisms regulating p38 activation during engagement of the Type I IFN receptor remain to be defined. We sought to identify the events that lead to activation of the p38 MAP kinase in response to Type I IFNs. Our data demonstrate that treatment of sensitive cell lines with IFNalpha results in activation of both MAP kinase kinase 3 (MKK3) and MAP kinase kinase 6 (MKK6). Such IFN-inducible activation of MKK3 and MKK6 is essential for downstream phosphorylation and activation of the p38 MAP kinase, as shown by studies using mouse embryonic fibroblasts (MEFs) with targeted disruption of the Mkk3 and Mkk6 genes (MKK3-/- MKK6-/-). Similarly, IFN-dependent activation of the downstream effectors of p38, MAPKAPK-2 and MAPKAPK-3, is not detectable in cells lacking Mkk3 and Mkk6, demonstrating that the function of these MAP kinase kinases is required for full activation of the p38 pathway. To define the functional relevance of MKK3/6 engagement in Type I IFN signaling, IFN-inducible gene transcription was evaluated in the MKK3/MKK6 double knock-out cells. IFNalpha- and IFNbeta-dependent transcription via either interferon-stimulated response element or IFNgamma activated site elements was defective in MKK3 -/-/MKK6 -/- MEFs in luciferase reporter assays. In addition, IFN-dependent induction of two genes known to be of importance in the generation of IFN responses, Isg15 and Irf-9, was diminished in the absence of Mkk3 and Mkk6. The effects of Mkk3 and Mkk6 on IFN-dependent transcription were unrelated to any effects on the phosphorylation and activation of STAT proteins, indicating the presence of a STAT-independent mechanism. Altogether, our findings demonstrate that MKK3 and MKK6 are rapidly activated during engagement of the Type I IFN receptor and play important roles in Type I IFN signaling and the generation of IFN responses.

Animals↗

PubMed2005

Role of protein kinase C-delta (PKC-delta) in the generation of the effects of IFN-alpha in chronic myelogenous leukemia cells.

OBJECTIVE: The mechanisms by which interferon alpha (IFN-alpha) induces antileukemic responses in chronic myelogenous leukemia (CML) cells are not known. We examined whether a member of the protein kinase C (PKC) family of proteins, PKC-delta, is activated during treatment of BCR-ABL cells with IFN-alpha and participates in the induction of interferon responses. METHODS: Immunoblots and immune complex kinase assays were performed to study the phosphorylation and activation of PKC-delta in response to IFN-alpha in CML-derived cell lines. The effects of pharmacological inhibition of PKC-delta on the suppressive effects of IFN-alpha on leukemic CFU-GM progenitors from CML patients were assessed by clonogenic assays in methylcellulose. RESULTS: IFN-alpha treatment of the sensitive CML-derived KT-1 cell line resulted in phosphorylation of PKC-delta and activation of its kinase domain. Such phosphorylation/activation of PKC-delta was required for phosphorylation of Stat1 on serine 727, as inhibition of PKC-delta activity blocked the IFN-alpha-dependent serine phosphorylation of Stat1 and IFN-alpha-inducible gene transcription. IFN-alpha treatment strongly inhibited leukemic CFU-GM progenitor colony formation from bone marrow or peripheral blood of patients with CML, and such inhibition was reversed by concomitant treatment of the cells with the PKC-delta pharmacologic inhibitor rottlerin. CONCLUSION: Taken altogether, our data demonstrate that PKC-delta plays a critical role in Type I IFN signaling in BCR-ABL expressing cells, acting as a serine kinase for Stat1, to regulate transcriptional activation of interferon-regulated genes and induction of antileukemic responses.

Cell Line, Tumor↗

PubMed2005

Mitogen-activated protein kinase pathways in interferon signaling.

There is recent evidence that in addition to the classic JAK/STAT pathways, mitogen-activated proteins (MAP) kinase pathways play important roles in Type I interferon signaling and the generation of interferon responses. In particular, the p38 MAP kinase cascade exerts positive regulatory roles on transciptional activation of interferon-sensitive genes (ISGs) and the induction of the antiproliferative and antiviral properties of interferons. In this chapter, methodologies to detect activation of p38 and various upstream and downstream effectors are provided. Also, the methodological approaches used to examine the role of this pathway on the regulation of hematopoiesis are discussed.

Animals↗

PubMed2004

EVI1 abrogates interferon-alpha response by selectively blocking PML induction.

EVI1 is an oncogene frequently associated with chronic and acute myeloid leukemia. In hematopoietic cells, EVI1 impairs several pathways including proliferation, differentiation, and apoptosis. Interferon-alpha (IFN-alpha) is a powerful cytokine that controls the immune response and limits the expansion of several tissues including bone marrow. These properties contribute to the effectiveness of IFN-alpha in the treatment of many neoplastic disorders especially chronic myeloid leukemia. We report here that in murine hematopoietic progenitors the expression of EVI1 completely abrogates the antiproliferative and apoptotic effects of IFN-alpha. EVI1 does not repress the JAK/STAT signaling pathway or the activation of many IFN-responsive genes. On the contrary, EVI1 prolongs the phosphorylation of STAT1 and the activation of an IFN-dependent reporter gene. However, EVI1 specifically represses the IFN-dependent induction of the tumor suppressor PML and blocks the apoptotic pathways activated by PML. We show that the position of the ISRE, which is located within the first exon of PML, is critical to block PML induction by IFN-alpha. The relocation of the ISRE to a position upstream of the transcription start site is sufficient to re-establish the response to IFN in the presence of EVI1. Our data suggest that stabilized STAT1 phosphorylation and prolonged binding of the STAT1 complex to the first exon could impair PML transcription and inhibit the activation of PML-dependent apoptotic pathways resulting in loss of IFN response. These results point to a novel mechanism utilized by an oncogene to escape normal cell response to growth-controlling cytokines.

Animals↗

PubMed2004

Activation of the p70 S6 kinase by all-trans-retinoic acid in acute promyelocytic leukemia cells.

Although the mechanisms by which all-trans-retinoic acid (RA) regulates gene transcription are well understood, very little is known on the signaling events regulating RA-dependent initiation of mRNA translation. We examined whether the mammalian target of rapamycin (mTOR)/p70 S6 kinase pathway is activated by RA. RA treatment of sensitive cell lines resulted in phosphorylation/activation of mTOR and downstream induction of p70 S6 kinase activity. Such phosphorylation/activation of p70 S6 kinase was inducible in primary acute promyelocytic leukemia (APL) blasts and RA-sensitive NB-4 cells, but was defective in an NB-4 variant cell line (NB-4.007/6) that is resistant to the biologic effects of RA. The RA-dependent activation of p70 S6 kinase was also phosphatidylinositol 3' kinase (PI3'K)-dependent, and resulted in downstream phosphorylation of the S6 ribosomal protein on Ser235/236 and Ser240/244, events important for initiation of translation for mRNAs with oligopyrimidine tracts in their 5' untranslated region. RA treatment of leukemia cells also resulted in an mTOR-mediated phosphorylation of the 4E-BP1 repressor of mRNA translation, to induce its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF-4E) complex. Altogether, these findings provide evidence for the existence of a novel RA-activated cellular pathway that regulates cap-dependent translation, and strongly suggest that this cascade plays a role in the induction of retinoid responses in APL cells.

Cell Differentiation↗

PubMed2004

Differential regulation of membrane type 1-matrix metalloproteinase activity by ERK 1/2- and p38 MAPK-modulated tissue inhibitor of metalloproteinases 2 expression controls transforming growth factor-beta1-induced pericellular collagenolysis.

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.

Biotinylation↗

PubMed2004

Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma.

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.

Calcium↗

PubMed2004

Engagement of protein kinase C-theta in interferon signaling in T-cells.

Protein kinase C-theta (PKC-theta) plays important roles in the activation and survival of lymphocytes and is the predominant PKC isoform expressed in T-cells. Interferons regulate T-cell function and activation, but the precise signaling mechanisms by which they mediate such effects have not been elucidated. We determined whether PKC-theta is engaged in interferon (INF) signaling in T-cells. Both Type I (alpha, beta) and Type II (gamma) IFNs induced phosphorylation of PKC-theta in human T-cell lines and primary human T-lymphocytes. Such phosphorylation of PKC-theta resulted in activation of its kinase domain, suggesting that this kinase plays a functional role in interferon signaling. Consistent with this, inhibition of PKC-theta protein expression using small interfering RNAs (siRNA) abrogated IFN-alpha- and IFN-gamma-dependent gene transcription via GAS elements. Similarly, blocking of PKC-theta kinase activity by overexpression of a dominant-negative PKC-theta mutant also blocked GAS-driven transcription, further demonstrating a requirement for PKC-theta in IFN-dependent transcriptional activation. The effects of PKC-theta on IFN-dependent gene transcription were not mediated by regulation of the IFN-activated STAT pathway, as siRNA-mediated PKC-theta knockdown had no effects on STAT1 phosphorylation and binding of STAT1-containing complexes to SIE/GAS elements. On the other hand, siRNA-mediated PKC-theta inhibition blocked phosphorylation/activation of MKK4, suggesting that interferon-dependent PKC-theta activation regulates downstream engagement of MAP kinase pathways. Altogether, these findings demonstrate that PKC-theta is an interferon-inducible kinase and strongly suggest that it plays an important role in the generation of interferon-responses in T-cells.

Cell Line↗

PubMed2004

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein.

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.

Bone Neoplasms↗