AIMP1 negatively regulates adipogenesis by inhibiting PPARγ.

Adipogenesis is known to be controlled by the concerted actions of transcription factors and co-regulators. However, little is known about the mechanism of regulation of the transcription factors that control adipogenesis. In addition, the adipogenic roles of translational factors remain unclear. Here, we show that aminoacyl tRNA synthetase-interacting multifunctional protein 1 (AIMP1, also known as p43), an auxiliary factor that is associated with a macromolecular tRNA synthetase complex, negatively regulates adipogenesis through a direct interaction with the DNA-binding domain of peroxisome proliferator-activated receptor γ (PPARγ). We found that AIMP1 expression increases during adipocyte differentiation. Adipogenesis is augmented in AIMP1-deficient cells, as compared with control cells. AIMP1 exhibits high affinity for active PPARγ and interacts with the DNA-binding domain of PPARγ, thereby inhibiting its transcriptional activity. Thus, AIMP1 appears to function as a novel inhibitor of PPARγ that regulates adipocyte differentiation by preventing the transcriptional activation of PPARγ.

Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction.

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression.

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

Fractionation of mature eosinophils in GATA-reporter transgenic mice.

Eosinophils contribute to the pathophysiology of allergic and infectious diseases, albeit their molecular functions remain unknown. Mature eosinophils are identified by their unique morphology and staining characteristics. However, it is difficult to fractionate living eosinophils by flow cytometry because these granulocytes express multiple cell surface markers that are shared by other cells of hematopoietic or non-hematopoietic origin. In this study, we describe a flow cytometry-based method to enumerate and fractionate eosinophils by developmental stages. To fractionate these cell types, we used transgenic mouse lines that express fluorescent proteins under control of the Gata1 gene hematopoietic regulatory region (Gata1-HRD), which is exclusively active in Gata1-expressing hematopoietic cells, including eosinophils. As expected, mature eosinophils were highly enriched in the fluorescent reporter-expressing subfraction of bone marrow myeloid cells that were negatively selected by using multiple antibodies against B220, CD4, CD8, Ter119, c-Kit and CD71. Cytochemical analyses of flow-sorted cells identified the cells in this fraction as eosinophils harboring eosinophilic granules. Additionally, expression of eosinophil-specific genes, for instance eosinophil enzymes and the IL-5 receptor alpha gene, were specifically detected in this fraction. The expression of these eosinophil-specific genes increased as the cells differentiated. This method for enrichment of bone marrow eosinophils is applicable to fractionation of eosinophils and bronchoalveolar lavage fluid from transgenic mice with atopic asthma. Thus, both pathological and developmental stages of eosinophils are efficiently fractionated by this flow cytometry-based method using Gata1-HRD transgenic reporter mice. This study, therefore, proposes a useful means to study the experimental allergic and inflammatory systems.

PEP-1-FK506BP inhibits alkali burn-induced corneal inflammation on the rat model of corneal alkali injury.

FK506 binding protein 12 (FK506BP) is a small peptide with a single FK506BP domain that is involved in suppression of immune response and reactive oxygen species. FK506BP has emerged as a potential drug target for several inflammatory diseases. Here, we examined the protective effects of directly applied cell permeable FK506BP (PEP-1-FK506BP) on corneal alkali burn injury (CAI). In the cornea, there was a significant decrease in the number of cells expressing pro-inflammation, apoptotic, and angiogenic factors such as TNF-α, COX-2, and VEGF. Both corneal opacity and corneal neovascularization (CNV) were significantly decreased in the PEP-1-FK506BP treated group. Our results showed that PEP-1-FK506BP can significantly inhibit alkali burn-induced corneal inflammation in rats, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors and inflammatory cytokines. These results suggest that PEP-1-FK506BP may be a potential therapeutic agent for CAI.

An arched micro-injector (ARCMI) for innocuous subretinal injection.

Several critical ocular diseases that can lead to blindness are due to retinal disorders. Subretinal drug delivery has been developed recently for the treatment of retinal disorders such as hemorrhage because of the specific ocular structure, namely, the blood retinal barrier (BRB). In the present study, we developed an Arched Micro-injector (ARCMI) for subretinal drug delivery with minimal retinal tissue damage. ARCMIs were fabricated using three major techniques: reverse drawing lithography, controlled air flow, and electroplating. In order to achieve minimal retinal tissue damage, ARCMIs were fabricated with specific features such as a 0.15 mm(-1) curvature, 45° tip bevel, 5 mm length, inner diameter of 40 µm, and an outer diameter of 100 µm. These specific features were optimized via in-vitro experiments in artificial ocular hemispherical structures and subretinal injection of indocyanine green in porcine eye ex-vivo. We confirmed that the ARCMI was capable of delivering ocular drugs by subretinal injection without unusual subretinal tissue damage, including hemorrhage.