The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.     

THOC5 regulates human osteoclastogenesis

Osteoclasts are bone resorbing cells that are responsible for physiological and pathological bone resorption. Macrophage colony stimulating factor (M-CSF) binds to the M-CSF receptor (c-FMS) and plays a key role in the differentiation and survival of macrophages and osteoclasts. THOC5, a member of the THO complex, has been shown to regulate hematopoiesis and M-CSF-induced macrophage differentiation. However, the role of THOC5 in osteoclasts remains unclear. Here, our study reveals a new role of THOC5 in osteoclast formation. We found that THOC5 shuttles between nucleus and cytoplasm in an M-CSF signaling dependent manner. THOC5 bound to FICD, a proteolytic cleavage product of c-FMS, and THOC5 facilitates the nuclear translocations of FICD. Decreased expression of THOC5 by siRNA-mediated knock down suppressed osteoclast differentiation, in part, by regulating RANK, a key receptor of osteoclasts. Mechanistically, knock down of THOC5 inhibited the expression of RANKL-induced FOS and NFATc1. Our findings highlight THOC5′s function as a positive regulator of osteoclasts.     

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Nuclear localization of the pre-mRNA associating protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP)

THOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50-137, amino acid numbers) binds to the N-terminal portion (1-199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.

Structured summary

MINT-6799962, MINT-6799973, MINT-6800005: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with THOC5 (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800108: FMIP (uniprotkb:Q13769) and THOC7 (uniprotkb: Q6I9Y2) co-localize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800052: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC1 (uniprotkb: Q96FV9) by anti tag coimmunoprecipitation (MI:0007)MINT-6800022: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with FMIP (uniprotkb:Q6DFL5) by pull down (MI:0096)MINT-6799989: THOC7 (uniprotkb:Q6I9Y2) binds (MI:0407) to FMIP (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800071, MINT-6800089: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC7 (uniprotkb:Q6I9Y2) and THOC1 (uniprotkb:Q96FV9) by anti tag coimmunoprecipitation (MI:0007)     

Comparison of in vitro and in vivo modulation of myelopoiesis by biological response modifiers

Summary In vitro growth and differentiation of granulocyte-macrophage progenitor cells (GM-CFU-C) requires colony-stimulating factors (CSF), and an in vivo role for CSF has also been proposed. Prostaglandins of the E series (PGE) have been reported to serve as negative feedback regulators of myelopoiesis. Here, we report evidence of augmented CSF secretion by mouse peritoneal Mo (macrophages) and bone marrow cells in vitro upon stimulation with various biological response modifiers (BRMs). Optimal induction of CSF secretion occurred after in vitro treatment of peritoneal Mo and mononuclear bone marrow cells with 50 g/ml poly ICLC (polyriboinosinic-polycytidylic acid poly-L-lysine), 5 g/ml lipopolysaccharide (LPS), or 500 U/ml interferon (IFN_,) for 2 days. The in vitro stimulation of CSF secretion was paralleled by an increase in PGE secretion by Mo and bone marrow cells. The PGE secretion could, however, be selectively blocked by preincubating the cells for 3 h with indomethacin (10^–7 Mol) leaving CFS production intact. In vivo treatment of mice with either maleic anhydride divinyl ether copolymer (MVE-2; 25 mg/kg) or poly ICLC (2 mg/kg) significantly increased levels of CSF in serum, as well as in culture supernatants of in vivo-treated peritoneal Mo and bone marrow cells. The increase in serum CSF levels and in secretion of CSF by peritoneal Mo and bone marrow cells was followed by a dose-dependent increase in GM-CFU-C, in nucleated bone marrow cells, and in peripheral blood leukocytes. The same BRMs also stimulated the secretion of PGE by in vivo-activated peritoneal Mo, but not by bone marrow cells. Pretreatment of the mice with indomethacin (4 mg/kg) almost completely suppressed PGE secretion by peritoneal Mo, but did not change the CSF secretion by peritoneal Mo or bone marrow cells and had no significant effect on bone marrow cellularity. Therefore, MVE-2 and poly ICLC, in addition to their immunomodulatory activity, can also have stimulatory effects on myelopoiesis, presumably mediated through secretion of CSFs. Protection and/or restoration of bone marrow function could thus either provide the opportunity for more extensive chemotherapy or could increase the number of Mo effector cells available for activation against tumor targets.