Multiple Post-transcriptional Mechanisms Research Articles

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

BRAF-V600E utilizes posttranscriptional mechanisms to amplify LPS-induced TNFα production in dendritic cells in a mouse model of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is an inflammatory disease characterized by abnormal dendritic cells (DCs) with hyperactive ERK signaling, called "LCH cells." Since DCs rely on ERK signaling to produce inflammatory molecules in response to pathogenic cues, we hypothesized that hyperactive ERK enhances DCs inflammatory responses. We specifically investigated TLR4-induced TNFα production in LCH cells by utilizing the BRAF-V600Efl/+ :CD11c-Cre mouse model of LCH, which hyperactivates ERK in DCs. We measured LPS-induced TNFα production both in vivo and in vitro using splenic CD11c+ cells and bone marrow-derived DCs with or without pharmacologic BRAFV600E inhibition. We observed a reversible increase in secreted TNFα and a partially reversible increase in TNFα protein per cell, despite a decrease in TLR4 signaling and Tnfa transcripts compared with controls. We examined ERK-driven, posttranscriptional mechanisms that contribute to TNFα production and secretion using biochemical and cellular assays. We identified a reversible increase in TACE activation, the enzyme required for TNFα secretion, and most strikingly, an increase in protein translation, including TNFα. Defining the translatome through polysome-bound RNA sequencing revealed up-regulated translation of the LPS-response program. These data suggest hyperactive ERK signaling utilizes multiple posttranscriptional mechanisms to amplify inflammatory responses in DCs, advancing our understanding of LCH and basic DC biology.

Biological implications of decapping: beyond bulk mRNA decay.

It is well established that mRNA steady-state levels do not directly correlate with transcription rate. This is attributed to the multiple post-transcriptional mechanisms, which control both mRNA turnover and translation within eukaryotic cells. One such mechanism is the removal of the 5' end cap structure of RNAs (decapping). This 5' cap plays a fundamental role in cellular functions related to mRNA processing, transport, translation, quality control, and decay, while its chemical modifications influence the fate of cytoplasmic mRNAs. Decapping is a highly controlled process, performed by multiple decapping enzymes, and regulated by complex cellular networks. In this review, we provide an updated synopsis of 5' end modifications and functions, and give an overview of mRNA decapping enzymes, presenting their enzymatic properties. Focusing on DCP2 decapping enzyme, a major component on the 5'-3' mRNA decay pathway, we describe cis-elements and trans-acting factors that affect its activity, substrate specificity, and cellular localization. Finally, we discuss current knowledge on the biological functions of mRNA decapping and decay factors, highlighting the major questions that remain to be addressed.

3080 – CHARACTERIZATION OF ALTERNATIVE SPLICING DYNAMICS IN T-CELL MATURATION AND T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Alternative splicing is one of the multiple post-transcriptional mechanisms that contribute to functional diversification in eukaryotic cells. Cancer-associated alternative splicing events have been reported in different tumor types, establishing new putative targets for diagnostic tools and therapies. Here, we explore the transcriptional program at exon resolution of T-Cell Acute Lymphoblastic Leukemia (T-ALL), an aggressive hematological malignancy characterized by the outgrowth of immature T-cells. To date, we have characterized the alternative splicing configuration of more than 120 RNA-Seq samples from four different T-ALL cohorts, normal T-cell maturation stages, and two cell lines, using vastdb. Exon skipping and intron retention are the most frequent events across all samples, with similar incidence in both cancer driver and non-driver genes. Interestingly, we observe that intron retention is significantly higher in immature and mature T-cells compared to leukemic cells. Conversely, exon skipping is higher in leukemic samples. These results suggest an impairment in the splicing machinery that might be driven by RNA binding proteins (RBPs) deregulation in leukemia. To further investigate that, we aim to discover which are the RBPs associated with isoform changes by constructing co-expression networks. The computational assessment of T-ALL specific isoforms will eventually help to improve the diagnosis, as well as the discovery of putative neoantigens targeted by CAR therapy.

Temperature Control of psaA Expression by PsaE and PsaF in Yersinia pestis.

PsaA, the subunit of the fimbria originally referred to as the "pH 6 antigen," is required for full virulence of Yersinia pestis during bubonic plague. The expression of psaA is dependent upon specific environmental signals, and while the signals (high temperature and acidic pH) are defined, the mechanisms underlying this regulation remain unclear. In the closely related species Yersinia pseudotuberculosis, psaA transcription requires two regulatory genes, psaE and psaF, and it is speculated that posttranscriptional regulation of PsaE and/or PsaF contributes to the regulation of psaA transcription. Few studies have examined the regulation of psaA expression in Y. pestis, and prior to this work, the roles of psaE and psaF in Y. pestis had not been defined. The data presented here show that both psaE and psaF are required for psaA transcription in Y. pestis and that the impact of temperature and pH is mediated through discrete posttranscriptional effects on PsaE and PsaF. By generating antibodies that recognize endogenous PsaE and PsaF, we determined that the levels of both proteins are impacted by temperature and pH. High temperature is required for psaE and psaF translation via discrete mechanisms mediated by the mRNA 5' untranslated region (UTR) upstream of each gene. Additionally, levels of PsaE and PsaF are impacted by pH. We show that PsaF enhances the stability of PsaE, and thus, both PsaE and PsaF are required for psaA transcription. Our data indicate that the environmental signals (temperature and pH) impact the expression of psaA by affecting the translation of psaE and psaF and the stability of PsaE and PsaF.IMPORTANCEY. pestis is a Gram-negative bacterial pathogen that causes bubonic plague. As a vector-borne pathogen, Y. pestis fluctuates between an arthropod vector (flea) and mammalian host. As such, Y. pestis must recognize environmental signals encountered within each host environment and respond by appropriately regulating gene expression. PsaA is a key Y. pestis mammalian virulence determinant that forms fimbriae. Our work provides evidence that Y. pestis utilizes multiple posttranscriptional mechanisms to regulate the levels of two PsaA regulatory proteins in response to both temperature and pH. This study offers insight into mechanisms that bacteria utilize to sense environmental cues and regulate the expression of determinants required for mammalian disease.

Identification and Characterization of the Physiological Gene Targets of the Essential Lytic Replicative Epstein-Barr Virus SM Protein.

Epstein-Barr virus (EBV) SM protein is an essential lytic cycle protein with multiple posttranscriptional mechanisms of action. SM binds RNA and increases accumulation of specific EBV transcripts. Previous studies using microarrays and PCR have shown that SM-null mutants fail to accumulate several lytic cycle mRNAs and proteins at wild-type levels. However, the complete effect of SM on the EBV transcriptome has been incompletely characterized. Here we precisely identify the effects of SM on all EBV transcripts by high-throughput RNA sequencing, quantitative PCR (qPCR), and Northern blotting. The effect of SM on EBV mRNAs was highly skewed and was most evident on 13 late genes, demonstrating why SM is essential for infectious EBV production. EBV DNA replication was also partially impaired in SM mutants, suggesting additional roles for SM in EBV DNA replication. While it has been suggested that SM specificity is based on recognition of either RNA sequence motifs or other sequence properties, no such unifying property of SM-responsive targets was discernible. The binding affinity of mRNAs for SM also did not correlate with SM responsiveness. These data suggest that while target RNA binding by SM may be required for its effect, specific activation by SM is due to differences in inherent properties of individual transcripts. We therefore propose a new model for the mechanism of action and specificity of SM and its homologs in other herpesviruses: that they bind many RNAs but only enhance accumulation of those that are intrinsically unstable and poorly expressed. This study examines the mechanism of action of EBV SM protein, which is essential for EBV replication and infectious virus production. Since SM protein is not similar to any cellular protein and has homologs in all other human herpesviruses, it has potential importance as a therapeutic target. Here we establish which EBV RNAs are most highly upregulated by SM, allowing us to understand why it is essential for EBV replication. By comparing and characterizing these RNA transcripts, we conclude that the mechanism of specific activity is unlikely to be based simply on preferential recognition of a target motif. Rather, SM binding to its target RNA may be necessary but not sufficient for enhancing accumulation of the RNA. Preferential effects of SM on its most responsive RNA targets may depend on other inherent characteristics of these specific mRNAs that require SM for efficient expression, such as RNA stability.

Methylxanthines Increase Expression of the Splicing Factor SRSF2 by Regulating Multiple Post-transcriptional Mechanisms

We have previously reported that the methylxanthine caffeine increases expression of the splicing factor SRSF2, the levels of which are normally controlled by a negative autoregulatory loop. In the present study we have investigated the mechanisms by which methylxanthines induce this aberrant overexpression. RT-PCR analyses suggested little impact of caffeine on SRSF2 total mRNA levels. Instead, caffeine induced changes in the levels of SRSF2 3' UTR splice variants. Although some of these variants were substrates for nonsense-medicated decay (NMD), and could potentially have been stabilized by caffeine-mediated inhibition of NMD, down-regulation of NMD by a genetic approach was not sufficient to reproduce the phenotype. Furthermore, cell-based assays demonstrated that some of the caffeine-induced variants were intrinsically more efficiently translated than others; the addition of caffeine increased the translational efficiency of most SRSF2 transcripts. MicroRNA array analyses revealed a significant caffeine-mediated decrease in the expression of two SRSF2-targeting miRs, both of which were shown to repress translation of specific SRSF2 splice variants. These data support a complex model whereby caffeine down-regulates SRSF2-targeting microRNAs, leading to an increase in SRSF2 translation, which in turn induces SRSF2 splicing. SRSF2 splice variants are then stabilized by caffeine-mediated NMD inhibition, breaking the normal negative feedback loop and allowing the aberrant increase in SRSF2 protein levels. These findings highlight the complexity of SRSF2 gene regulation, and suggest ways in which SRSF2 expression may be dysregulated in disease.

Novel role of Na+,K+-ATPase ligands in regulating cytokines mRNA stability by HuR signalosome and the underlying pathophysiologic relevance

Increasing evidence demonstrated that Na + ,K + -ATPase ligands, also called as cardiotonic steroids, are hormone-like immunoregulators, because endogenous Na + ,K + -ATPase ligands are frequently detected in inflammatory-related diseases, moreover, Na + ,K + -ATPase ligands regulate multiple aspects of immune responses. One of prominent roles of Na + ,K + -ATPase ligands in regulating immunity is their abilities of modulating cytokines expression. Na + ,K + -ATPase ligands can either upregulate or downregulate IL-1β, TNF-α, IL6, or iNOS expressions in different model system, however, all of those studies pointed to transcriptional upregulation. In our recent studies, we proposed for the first time that Na + ,K + -ATPase ligands are capable of regulating cytokines mRNA stability by integrating multiple posttranscriptional mechanisms, including human antigen R (HuR) translocation, generation of miR181s, and formation of stress granules. These mechanisms did not function alone, but act in a synergistic or an antagonistic manner to fine-tune the cytokines expression, HuR nuclear export, however, forms signalosome and plays a core role among these processes. By taking advantage of these posttranscriptional mechanisms, Na + ,K + -ATPase ligands stabilized cyclooxygenase-2 mRNA stability in lung epithelial cells and induced acute lung injury. In monocytes, ouabain-induced HuR export competed with miR181s on the shared target of TNF-α, also triggered stress granules formation and recruited TNF-α mRNA into it for protection, thereby stabilizing TNF-α mRNA and reversing sepsis-induced immunoparalysis, both in vitro and in vivo. Besides in immune-related diseases, HuR also regulated a variety of pro-oncogenes and anti-oncogenes expressions in cancer cells, which determined the cancer cells sensitivity towards Na + ,K + -ATPase ligands or other chemotherapeutic drugs. In sum, HuR emerges as a very important signaling molecule in Na + ,K + -ATPase ligands-mediated effects, which opens new avenues for understanding of the pathophysiologic and pharmacological activities of Na + ,K + -ATPase ligands. Identification of the components of HuR signalosome will offer more novel targets and biomarkers for diseases therapy.

Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium

HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements and related motifs present in the 3'untranslated region (UTR) of mRNAs. We postulate that HuR critically regulates the epithelial response by associating with multiple ARE-bearing, functionally related inflammatory transcripts. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNF-α plus IFN-γ, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein complexes from resting and cytokine-treated cells were immunoprecipitated using anti-HuR and isotype-control Ab, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1, and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control immunoprecipitation. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene ribonucleoprotein-immunoprecipitation and shown to be 3'UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic. Conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. HuR-mediated regulation in airway epithelium appears broader than previously appreciated, coordinating numerous inflammatory genes through multiple posttranscriptional mechanisms.

Novel Proteolytic Processing of the Ectodomain of the Zinc Transporter ZIP4 (SLC39A4) during Zinc Deficiency Is Inhibited by Acrodermatitis Enteropathica Mutations

The zinc transporter ZIP4 (SLC39A4) is mutated in humans with the rare, autosomal recessive genetic disease acrodermatitis enteropathica. In mice, this gene is essential during early embryonic development. ZIP4 is dynamically regulated by multiple posttranscriptional mechanisms, and studies of mouse ZIP4 reported herein reveal that the ectodomain, the extracellular amino-terminal half of the protein, is proteolytically removed during prolonged zinc deficiency while the remaining eight-transmembrane carboxyl-terminal half of the protein is accumulated on the plasma membrane as an abundant form of ZIP4. This novel ZIP4 processing occurs in vivo in the intestine and visceral endoderm, in mouse Hepa cells that express the endogenous Slc39a4 gene and in transfected MDCK and CaCo2 cells, but not HEK293 cells. In transfected MDCK and CaCo2 cells, the ectodomain accumulated and remained associated with membranes when zinc was deficient. ZIP4 cleavage was attenuated by inhibitors of endocytosis, which suggests that the processed protein is recycled back to the plasma membrane and that the ectodomain may be internalized. Ectodomain cleavage is inhibited by acrodermatitis enteropathica mutations near a predicted metalloproteinase cleavage site which is also essential for proper ectodomain cleavage, and overexpression of processed ZIP4 or ZIP4 with ectodomain truncations rendered the mouse Mt1 gene hypersensitive to zinc. These finding suggest that the processing of ZIP4 may represent a significant regulatory mechanism controlling its function.

Regulation and function of Zip4, the acrodermatitis enteropathica gene

The SLC39A (solute carrier 39A) [ZIP (Zrt-Irt-like protein)] family consists of 14 members which are thought to control zinc uptake into the cytoplasm. Among these, ZIP4 is known to be particularly important for zinc homoeostasis. Mutations in this gene cause acrodermatitis enteropathica, a rare recessive-lethal human genetic disorder. In the present paper, our studies of the regulation and function of the mouse Zip4 gene are briefly reviewed. Mouse Zip4 is expressed at highest levels in tissues involved in absorption of dietary or maternal zinc, and the gene and protein are dynamically regulated by multiple post-transcriptional mechanisms in response to zinc availability. ZIP4 accumulates at the apical surface of enterocytes and endoderm cells when zinc is deficient, because of increased stability of the mRNA and stabilization of the protein. In contrast, when zinc is replenished, the mRNA is destabilized and the protein is internalized and degraded rapidly. The critical importance of ZIP4 in zinc homoeostasis is revealed in mice with targeted deletions of this gene. Homozygous Zip4-knockout embryos die during early morphogenesis and heterozygous offspring are significantly underrepresented and display an array of developmental defects, including exencephalia, anophthalmia and severe growth retardation. Mice heterozygous for Zip4-knockout are hypersensitive to zinc deficiency, which suggests that humans heterozygous for this gene may also be very sensitive to zinc deficiency.

Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) Suppresses Activity of PI3K/Akt Pathway and Regulates Expression of p27 (Kip1) and c-myc, DAP5 and Willm's Tumor (WT1) in Acute Myeloid Leukemia.

Programmed cell death 4 (PDCD4) is a recently identied novel tumor suppressor protein that inhibits cap-dependent mRNA translation. PDCD4 inhibits tumor promoter incuced carcinogenesis and transformation by suppressing the helicase activity of eIF4A, leading to translational inhibition. Recently we found that PDCD4 is required for all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of acute promyelocytic leukemia (APL) cells (Ozpolat&Akar et al, Mol Cancer Res, in press), type of acute myeloid leukemia characterized by a t(15;17) and a differentiation block. Here we investigated the downstream mediators or targets of PDCD4 in leukemia cell differentiation. ATRA is currently used as a first line standard treatment in APL. Recently, we reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms that involve suppression of translation initiation, a rate limiting step of protein synthesis (Harris&Ozpolat et al, Blood , 104 (5) 2004). We found that ATRA treatment induced PDCD4 expression in NB4 APL, HL60 AML, primary APL patient leukemia cells and normal human CD34+ bone marrow progenitors cells during granulocytic differentiation. However, ATRA/maturation resistant NB4.R1 and HL60R cells failed to express and translocate PDCD4 into nucleus after ATRA treatment. To identify downstream targets of PDCD4 we knock downed PDCD4 expression by siRNA and examined changes in the expression of target proteins that are known to be regulated by ATRA by Western blot analysis in NB4 cells. We found that PDCD4 represses c-myc, and WT1 expression however, it is required for the expression of DAP5 (death associated protein 5), and cyclin dependent kinase inhibitor p27KIP1, and but not c-jun, p21Cip1, and tissue transglutaminase (TG2). Inhibition of PDCD4 by siRNA resulted in upregulation of phospho-P70S6K, suggesting that PDCD4 inhibits activity of PI3K/Akt pathway. RT-PCR analysis revealed that mRNA of these proteins did not change suggesting that PDCD4 tumor suppressor protein regulates expression of these important cellular proteins at translational level and suppresses PI3K/Akt pathway. Furthermore we rapamycin, a specific mTOR inhibitor currently in clinical trials in AML, induced a marked expression of PDCD4, which regulates c-myc and p27 Kip1, revealing a novel mechanism of action of rapamycin, providing new rationale for targeting translational pathways as a therapeutic intervention in the treatment of AML. Overall, data suggest that PDCD4 regulates expression of critical cellular proteins involved in differentiation of leukemia cells and PDCD4 mediated translational control may be an important regulatory mechanism for regulation of gene expression.

P38 MAPK Signaling Mediates Retinoic Acid-Induced Expression of a Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) in Acute Promyelocytic Leukemia Cells.

Programmed-cell-death-4 (PDCD4) is a novel tumor suppressor protein that suppresses tumor promoter-induced neoplastic transformation. PDCD4 specifically inhibits the helicase activity of eukaryotic translation initiation factor 4A (eIF4A) and translation initiation and cap-dependent mRNA translation in vitro and in vivo. Loss or underexpression of PDCD4 is associated with carcinogenesis and chemoresistance in solid tumors. The role and regulation of PDCD4 in the the hematopoietic system and myeloid leukemia cells are not known. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation (Harris et al, Blood, 104 (5) 2004). Therefore, in this study, we investigated the expression and regulation of PDCD4 during myeloid cell differentiation. All-trans-retinoic acid (ATRA) induces terminal differentiation in acute myeloid leukemia (AML) and promyelocytic (APL) cells, a well established model for myeloid cell differentiation. We found that treatment of HL60 (M2 type AML) and NB4 APL (M3 type AML) cells with ATRA (1 mM) induced PDCD4 protein and mRNA expression during granulocytic differentiation detected by western blot and RT-PCR analysis, respectively. We also demonstrated that inhibition of PDCD4 by siRNA reduced granulocytic differentiation induced by ATRA, suggesting that PDCD4 plays a role in granuliocytic differentiation. To determine mechanisms regulating PDCD4 we investigated the role of pP38 MAPK (Mitogen activated protein kinase) in reugulation of PDCD4 expression. ATRA induced PDCD4 expression correlated with activation of p38 MAPK (Mitogen Activated Protein Kinase) pathway in NB4 cells. To test the hypothesis that p38 MAPK signaling pathway mediates retinoic acid induced PDCD4 expression we treated cells with a specific p38 MAPK inhibitor, SB203580, ATRA or combination with ATRA. We observed that p38 inhibitor inhibited ATRA-induced expression of PDCD4 in NB4 cells. Basal level of PDCD4 expression was also markedly downregulated in the presence of p38 inhibitor when compared to untreated control cells, suggesting that p38 pathway is involved in ATRA-dependent and independent PDCD4 expression. Currently we are investigating whether inhibition of p38 by small interfering RNA (siRNA) will prevent expression of ATRA induced PDCD4 in APL cells. We are also trying to identify whether ATF2 transcription factor, a downstream of p38, is involved in PDCD4 expression. p38-mediated induction of PDCD4 pathway reveals a novel mechanism of PDCD4 regulation and ATRA action, providing a new insight into understanding terminal differentiation of myeloid cells. Better understanding the role of PDCD4 and posttranscriptional control of gene expression may offer targets for the differentiation therapy and chemo preventive strategies.

PKCδ Regulates Eukaryotic Initiation Factor eIF2α through PKR during Retinoic Acid-Induced Myeloid Cell Differentiation.

Dysregulation of mRNA translation can contribute to malignant transformation. Translation initiation is a rate limiting step of mRNA translation and protein synthesis and plays a critical role in regulation of cell growth, proliferation and differentiation. We previously reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms during terminal cell differentiation detected by proteomic analysis (Harris et al, Blood, 104 (5) 2004). Here we investigated the regulation of translation initiation and the role of eIF2α during terminal differentiation of myeloid leukemia cells. We found that ATRA and other granulocytic differentiation inducing agents, such as dimethyl sulfoxide (DMSO), arsenic trioxide (ATO) induce phosphorylation of eIF2α on serine 51 in promyelocytic leukemia (NB4) cells, indicating the suppression of translation initiation. However, monocytic/macrophagic differentiation of NB4 cells by phorbol 12-myristate 13-acetate (phorbol ester, PMA), or by ATRA in U937 and THP-1 myelomonoblastic myeloid leukemia (AML) cells, was not accompanied with induction of eIF2α phosphorylation. ATRA, ATO or DMSO-induced granulocytic differentiation closely correlated with induction of expression and phosphorylation/activation of protein kinase C-delta (PKCδ) on threonin 505 and serine 643 in NB4 cells. The specific PKCδ inhibitor, rottlerin, markedly inhibited ATRA-induced expression and phosphorylation (serin 51) of eIF2a in NB4 cells. Rottlerin reduced phosphorylation of eIF2α expression not only in the leukemia cells but also in solid tumor cells such as breast (MCF7) and pancreatic (Panc28) cancer cells. Because protein kinase R (PKR) has been shown to inhibit mRNA translation by inducing phosphorylation of eIF2α, we also examined whether this pathway is involved in ATRA-induced phosphorylation of eIF2α and whether it is downstream of PKCδ. We observed that ATRA induces expression and phosphorylation/activation of PKR in NB4 cells. Rottlerin inhibited ATRA-induced expression and activity of PKR , suggesting that activity of PKR is regulated by PKCδ in response to ATRA in NB4 cells. Overall, our data suggest that retinoic acid suppresses translation initiation through PKCδ/PKR/eIF2α pathway during granulocytic but not monocytic differentiation of acute myeloid leukemia cells. These results revealed a novel role of ATRA in granulocytic cell differentiation of myeloid cells. Because malignant cells usually have hyperactivated mRNA translation, targeting translational factors/regulators of initiation may offer new strategies for the treatment of myeloid leukemia cells.

All-trans-Retinoic acid (ATRA) and Arsenic Trioxide-Induced Expression and Regulation of Programmed Cell Death 4 (PDCD4) in Acute Promyelocytic Leukemia.

All-trans-retinoic acid (ATRA) induces growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia (APL) characterized by t(15;17), which leads to expression of PML-RARa and differentiation arrest. ATRA treatment alone results in complete remission (CR) about 90% of APL patients. However these remissions are transient and APL patients commonly become resistant to ATRA therapy. Recently, arsenic trioxide, As(2)O(3), was proven to be highly effective in inducing CRs not only in relapsed after ATRA and primary APL patients. Despite the well documented clinical efficacy ATRA and As(2)O(3) in APL, precise downstream molecular mechanisms of action of these agents and the molecular mechanisms responsible for the resistance largely remain unknown. Recently, employing comprehensive proteomics methods we studied molecular mechanisms of ATRA-induced growth inhibition in APL cells. We reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms involved in translation initiation and elongation phases (Harris &Ozpolat et al, Blood, 104 (5) 2004). We also demonstrated that ATRA inhibits expression translation initiation factors including IF2, eIF4AI, eIF4G, eIF5, eIF6 but upregulates expression of translation inhibitor DAP5/p97/NAT1 in APL cells. Translational control of gene expression has been identified as an important regulatory mechanism for gene products involved in regulation of cell proliferation, differentiation and apoptosis. Programmed cell death 4(PDCD4) inhibits cap-dependent translation and exerts transformation-suppressing activity by inhibiting the helicase activity of eIF4A. . Here we investigated whether PDCD4 plays a role in ATRA-induced growth inhibition and terminal differentiation by mediating translational suppression. We found for the first time that ATRA (1 mM) and As(2)O(3) ( 0.4 mM and 2 mM) induced PDCD4 expression at mRNA and protein level detected by RT-PCR and western blotting analyses, respectively, after 24 h of treatment in NB4 cells. As(2)O(3) induced maximum PDCD4 protein expression at 72 h of treatment in NB4 cells ATRA induced PDCD4 mRNA expression in ATRA responsive human acute myeloid leukemia cells (HL-60) but not in ATRA-resistant HL60 cells (HL-60R), which express a point mutation in ATRA binding domain of RARa, suggesting PDCD4 expression is mediated through retinoid receptor alpha in HL-60 leukemia cells. Overall data suggest that translational control may play a role in ATRA-induced differentiation and As(2)O(3)-induced effects. We are currently determining whether other retinoid receptors are involved in ATRA-induced expression of PDCD4 and testing the hypothesis that whether PDCD4-mediated translational suppression is critical to ATRA-induced APL cell differentiation. Activation of these pathways that lead to translational suppression reveals a novel mechanism of ATRA action and provide novel insights into ATRA-induced differentiation program in APL cells.Better understanding of posttranscriptional control of gene expression may offer targets for the differentiation therapy and chemo preventive strategies.

Autoinhibitory domain fragment of endothelial NOS enhances pulmonary artery vasorelaxation by the NO-cGMP pathway.

Catalytic activity of eNOS is regulated by multiple posttranscriptional mechanisms, including a 40-amino acid (604-643) autoinhibitory domain (AID) located in the reductase domain of the eNOS protein. We examined whether an exogenous synthetic AID, an 11-amino acid (626-636) fragment of AID (AAF), or scrambled AAF (AAF-SR), enhanced eNOS activity and NO-cGMP-mediated vasorelaxation using pulmonary artery (PA) endothelial/smooth muscle cell (PAEC/PASM) coculture, isolated PA segment, and isolated lung perfusion models. Incubation of isolated total membrane fraction of PAEC with AID or AAF resulted in concentration-dependent loss of eNOS activity. In contrast, incubation of intact PAEC with AID or AAF but not AAF-SR caused concentration- and time-dependent activation of eNOS. Because AID and AAF had similar effects on activation of eNOS, AAF and AAF-SR were used for further evaluation. Although AAF stimulation increased catalytic activity of PKC-alpha in PAEC, AAF-mediated activation of eNOS was independent of phosphorylation of Ser1177 or Thr495 and/or expression of eNOS protein. AAF stimulation of PAEC increased NO and cGMP production, which were attenuated by pretreatment with the eNOS inhibitor l-NAME. AAF caused time-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PA segments, and this response was attenuated by l-NAME. AAF, but not AAF-SR, also caused vasorelaxation in an ex vivo isolated mouse lung perfusion model precontracted with U-46619. Incubation with fluorescence-labeled AAF demonstrated translocation of AAF in PAEC in culture, isolated PA, and isolated intact lungs. These results demonstrate that AAF-stimulated vasodilation is mediated via activation of eNOS and enhanced NO-cGMP production in PA and intact lung.

Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells.

Inflammation likely has a role in the early genesis of certain malignancies. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms. Here, we engineered a transgenic mouse to overexpress IL-15 by eliminating these posttranscriptional checkpoints. IL-15 transgenic mice have early expansions in natural killer (NK) and CD8+ T lymphocytes. Later, these mice develop fatal lymphocytic leukemia with a T-NK phenotype. These data provide novel evidence that leukemia, like certain other cancers, can arise as the result of chronic stimulation by a proinflammatory cytokine.

Fatal Leukemia in Interleukin-15 Transgenic Mice

ABSTRACTThe role of inflammation in the early genesis of certain malignancies has recently been appreciated. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms, suggesting that inappropriate expression of IL-15 may be detrimental to the host. We recently engineered a transgenic mouse in which the normal posttranscriptional control of IL-15 is eliminated, thereby overexpressing the murine IL-15 protein. IL-15 transgenic mice have early expansions in NK and CD8+ T lymphocytes and later develop fatal lymphocytic leukemia with a T-NK phenotype. This article recapitulates the phenotype of these IL-15 transgenic mice and discusses the utility of this model as a tool to further our understanding of leukemogenesis.

Sialomucin Complex (Rat Muc4) Is Regulated by Transforming Growth Factor β in Mammary Gland by a Novel Post-translational Mechanism

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (for ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, produced from a single gene and precursor. SMC expression is tightly regulated in mammary gland; the level in lactating mammary gland is about 100-fold that in virgin gland. In rat mammary epithelial cells, SMC is post-transcriptionally regulated by Matrigel by inhibition of SMC precursor synthesis. SMC is also post-transcriptionally regulated by transforming growth factor-beta (TGFbeta). The repression of SMC expression by TGFbeta is rapid, is independent of TGFbeta-induced cell cycle arrest, and does not require new protein synthesis. Unlike Matrigel, TGFbeta does not reduce SMC protein synthesis, as SMC precursor accumulation is equivalent in TGFbeta-treated and untreated cells. Instead, SMC precursor in TGFbeta-treated cells is more persistent and does not become processed as rapidly into mature ASGP-1 and ASGP-2, indicating that TGFbeta disrupts processing of SMC precursor. These results indicate that SMC, a product of normal mammary gland and milk, is regulated by TGFbeta by a novel post-translational mechanism. Thus, SMC is regulated by multiple post-transcriptional mechanisms, which serve to repress potential deleterious effects of overexpression.