Calin Dan Dumitru

Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia

Micro-RNAs (miR genes) are a large family of highly conserved noncoding genes thought to be involved in temporal and tissue-specific gene regulation. MiRs are transcribed as short hairpin precursors (≈70 nt) and are processed into active 21- to 22-nt RNAs by Dicer, a ribonuclease that recognizes target mRNAs via base-pairing interactions. Here we show that miR15 and miR16 are located at chromosome 13q14, a region deleted in more than half of B cell chronic lymphocytic leukemias (B-CLL). Detailed deletion and expression analysis shows that miR15 and miR16 are located within a 30-kb region of loss in CLL, and that both genes are deleted or down-regulated in the majority (≈68%) of CLL cases.

Modulation of miR-155 and miR-125b Levels following Lipopolysaccharide/TNF-α Stimulation and Their Possible Roles in Regulating the Response to Endotoxin Shock

We report here that miR-155 and miR-125b play a role in innate immune response. LPS stimulation of mouse Raw 264.7 macrophages resulted in the up-regulation of miR-155 and down-regulation of miR-125b levels. The same changes also occurred when C57BL/6 mice were i.p. injected with LPS. Furthermore, the levels of miR-155 and miR-125b in Raw 264.7 cells displayed oscillatory changes in response to TNF-α. These changes were impaired by pretreating the cells with the proteasome inhibitor MG-132, suggesting that these two microRNAs (miRNAs) may be at least transiently under the direct control of NF-κB transcriptional activity. We show that miR-155 most probably directly targets transcript coding for several proteins involved in LPS signaling such as the Fas-associated death domain protein (FADD), IκB kinase ε (IKKε), and the receptor (TNFR superfamily)-interacting serine-threonine kinase 1 (Ripk1) while enhancing TNF-α translation. In contrast, miR-125b targets the 3′-untranslated region of TNF-α transcripts; therefore, its down-regulation in response to LPS may be required for proper TNF-α production. Finally, Eμ-miR-155 transgenic mice produced higher levels of TNF-α when exposed to LPS and were hypersensitive to LPS/d-galactosamine-induced septic shock. Altogether, our data suggest that the LPS/TNF-α-dependent regulation of miR-155 and miR-125b may be implicated in the response to endotoxin shock, thus offering new targets for drug design.

TNF-α Induction by LPS Is Regulated Posttranscriptionally via a Tpl2/ERK-Dependent Pathway

Abstract Tpl 2 knockout mice produce low levels of TNF-α when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-κB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-α induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-α mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-α. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-α mRNA from the nucleus to the cytoplasm.

Induction of COX-2 by LPS in macrophages is regulated by Tpl2-dependent CREB activation signals

Macrophage activation by bacterial lipopolysaccharide (LPS) promotes the secretion of pro‐inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β), and of secondary mediators, such as leukotrienes and prostaglandins (PGs). Mice lacking the gene encoding the serine/threonine protein kinase Tpl2/Cot produce low levels of TNF‐α in response to LPS because of an ERK‐dependent post‐transcriptional defect, and they are resistant to LPS/D‐galactosamine‐induced endotoxin shock. In this study we demonstrate that prostaglandin E2 and its regulatory enzyme, COX‐2, are also targets of Tpl2‐transduced LPS signals in bone marrow‐derived mouse macrophages. Thus, LPS‐stimulated Tpl2−/− macrophages express low levels of COX‐2 and PGE2, compared with wild‐type Tpl2+/+ cells. The ability of Tpl2 to regulate COX‐2 expression depends on ERK signals that activate p90Rsk and Msk1, which in turn phosphorylate CREB, a key regulator of COX‐2 transcription. These data identify physiological targets of Tpl2 signaling downstream of ERK and further implicate Tpl2 in the pathophysiology of inflammation.

Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40

Macrophages from Tpl2 knockout (Tpl2−/−) mice exhibit a defect in ERK activation by lipopolysaccharide (LPS). This impairs the nucleocytoplasmic transport of the tumor necrosis factor α (TNF‐α) mRNA and prevents the induction of TNF‐α by LPS. As a result, Tpl2−/− mice are resistant to LPS/D‐galactosamine‐induced shock. We demonstrate that Tpl2 is essential for ERK signals transduced by members of the TNF receptor superfamily, such as CD40 and the TNF receptor 1. Thus, ERK activation was impaired in Tpl2−/− B cells and macrophages stimulated with agonistic CD40 antibody or TNF‐α, whereas the induction of other mitogen‐activated protein kinases, such as JNK and p38, and the activation of NF‐κB were unaffected. Tpl2 was recruited to a CD40/TRAF6 complex in response to CD40 stimulation. Moreover, TRAF6, which when overexpressed activates ERK, failed to do so in Tpl2−/− cells. The selective signaling defect resulting from the inactivation of Tpl2 allowed us to demonstrate that CD40‐mediated ERK activation contributes to immunoglobulin production but is not essential for B‐cell proliferation.

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the “paradoxical” Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.