Hiroko Kishikawa

The Cell Type-Specific Expression of the Murine IL-13 Gene Is Regulated by GATA-3

IL-13, a Th2 cell-specific cytokine, is a major effector molecule mediating several pathological features of allergic asthma. However, the transcriptional regulation of the IL-13 gene remains unclear. Here we demonstrate, by using intracellular cytokine staining, that IL-13 is not always coexpressed with other Th2 cytokines in normal Th cells on a single cell basis. In addition, we identified and cloned a minimal inducible and cell type-specific promoter of the murine IL-13 gene. The cell type specificity of the minimal IL-13 promoter is mediated by a functionally critical GATA-3 site that binds endogenous GATA-3 proteins, whereas the induction by PMA/ionomycin is mediated by distinct cis-acting elements. Furthermore, by expressing GATA-3 in wild-type and c-maf transgenic Th1 cells, we demonstrate that the expression of IL-13 is regulated by a mechanism distinct from that regulating the expression of IL-4, and that the expression of Th1 and Th2 cytokine genes does not have to be mutually exclusive in effector Th cells.

ROG, Repressor of GATA, Regulates the Expression of Cytokine Genes

GATA-3 is a T cell-specific transcription factor and is essential for the development of the T cell lineage. Recently, it was shown that the expression of GATA-3 is further induced in CD4+ helper T cells upon differentiation into type 2 but not type 1 effector cells. Here, we report the molecular cloning of a GATA-3 interacting protein, repressor of GATA (ROG). ROG is a lymphoid-specific gene and is rapidly induced in Th cells upon stimulation with anti-CD3. In in vitro assays, ROG represses the GATA-3-induced transactivation. Furthermore, overexpression of ROG in Th clones inhibits the production of Th cytokines. Taken together, our results suggest that ROG might play a critical role in regulating the differentiation and activation of Th cells.

Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis

Heterozygous missense mutations in the coding region of angiogenin (ANG), an angiogenic ribonuclease, have been reported in amyotrophic lateral sclerosis (ALS) patients. However, the role of ANG in motor neuron physiology and the functional consequences of these mutations are unknown. We searched for new mutations and sought to define the functional consequences of these mutations.

Transcriptional regulation of th2 differentiation by inducible costimulator.

Helper T (Th) cell differentiation is accompanied by complex transcriptional changes. Although costimulatory receptors are important in Th differentiation, the underlying mechanisms are poorly understood. Here we examine the transcriptional mechanisms by which ICOS regulates Th2 differentiation and selective IL-4 expression by effector T cells. We found impaired expression of c-Maf transcription factor functionally associated with the IL-4 defect in ICOS(-/-) cells. c-Maf expression in effector cells was regulated by IL-4 levels during Th differentiation. ICOS costimulation potentiated the T cell receptor (TcR)-mediated initial IL-4 production, possibly through the enhancement of NFATc1 expression. These data indicate that ICOS, by enhancing TcR signals at an early stage of T cell activation, regulates IL-4 transcription and T cell function in effector cells.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis

Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD.

Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123

Abstract Kishikawa, H., Wang, K., Adelstein, S. J. and Kassis, A. I. Inhibitory and Stimulatory Bystander Effects are Differentially Induced by Iodine-125 and Iodine-123. Radiat. Res. 165, 688–694 (2006). The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated 125I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765–13770, 2002). We have repeated these studies using cells exposed to lethal doses of 123I, an Auger electron emitter whose emission spectrum is identical to that of 125I, and report herein that the decay of 123I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects (125I) and stimulatory bystander effects (123I) are obtained in vitro. Moreover, supernatants from cultures with 125I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with 123I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose–response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all “calculated” dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.

Angiogenin-stimulated rRNA transcription is essential for initiation and survival of AKT-induced prostate intraepithelial neoplasia.

Angiogenin (ANG), originally identified as an angiogenic ribonuclease, has recently been shown to play a direct role in prostate cancer cell proliferation by mediating rRNA transcription. ANG is up-regulated in human prostate cancer and is the most significantly up-regulated gene in AKT-driven prostate intraepithelial neoplasia (PIN) in mice. Enhanced cell proliferation in the PIN lesions requires increased ribosome biogenesis, a multistep process involving an orchestrated production of ribosomal proteins and rRNA. AKT is known to enhance ribosomal protein production through the mammalian target of rapamycin pathway. However, it was unknown how rRNA is proportionally increased. Here, we report that ANG is essential for AKT-driven PIN formation and survival. We showed that up-regulation of ANG in the AKT-overexpressing mouse prostates is an early and lasting event. It occurs before PIN initiation and lasts beyond PIN is fully developed. Knocking down ANG expression by intraprostate injection of lentivirus-mediated ANG-specific small interfering RNA prevents AKT-induced PIN formation without affecting AKT expression and its signaling through the mammalian target of rapamycin pathway. Neomycin, an aminoglycoside that blocks nuclear translocation of ANG, and N65828, a small-molecule enzymatic inhibitor of the ribonucleolytic activity of ANG, both prevent AKT-induced PIN formation and reverse established PIN. They also decrease nucleolar organizer region, restore cell size, and normalize luminal architectures of the prostate despite continuous activation of AKT. All three types of the ANG inhibitor suppress rRNA transcription of the prostate luminal epithelial cells and inhibit AKT-induced PIN, indicating an essential role of ANG in AKT-mediated cell proliferation and survival. (Mol Cancer Res 2009;7(3):415–24)

Targeting angiogenin in therapy of amyotropic lateral sclerosis

Background: Missense heterozygous mutations in the coding region of angiogenin (ANG) gene, encoding a 14 kDa angiogenic RNase, were recently found in patients of amyotropic lateral sclerosis (ALS). Functional analyses have shown that these are loss-of-function mutations, implying that angiogenin deficiency is associated with ALS pathogenesis and that increasing ANG expression or angiogenin activity could be a novel approach for ALS therapy. Objective: Review the evidence showing the involvement of angiogenin in motor neuron physiology and function, and provide a rationale for targeting angiogenin in ALS therapy. Methods: Review the current understanding of the mechanism of angiogenin action in connection with ALS genetics, pathogenesis and therapy. Conclusion: ANG is the first gene whose loss-of-function mutations are associated with ALS pathogenesis. Therapeutic modulation of angiogenin level and activity in the spinal cord, either by systemic delivery of angiogenin protein or through retrograde transport of ANG-encoding viral particles, may be beneficial for ALS patients.

Angiogenin prevents serum withdrawal-induced apoptosis of P19 embryonal carcinoma cells.

Angiogenin is a 14 kDa protein originally identified as an angiogenic protein. Recent development has shown that angiogenin acts on both endothelial cells and neuronal cells. Loss‐of‐function mutations in the coding region of the ANG gene have recently been identified in patients with amyotrophic lateral sclerosis. Angiogenin has been shown to control motor neuron survival and protect neurons from apoptosis under various stress conditions. In this article, we characterize the anti‐apoptotic activity of angiogenin in pluripotent P19 mouse embryonal carcinoma cells. Angiogenin prevents serum withdrawal‐induced apoptosis. Angiogenin upregulates anti‐apoptotic genes, including Bag1, Bcl‐2, Hells, Nf‐κb and Ripk1, and downregulates pro‐apoptotic genes, such as Bak1, Tnf, Tnfr, Traf1 and Trp63. Knockdown of Bcl‐2 largely abolishes the anti‐apoptotic activity of angiogenin, whereas the inhibition of Nf‐κb activity results in a partial, but significant, inhibition of the protective activity of angiogenin. Thus, angiogenin prevents stress‐induced cell death through both the Bcl‐2 and Nf‐κb pathways.

Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress

Altered RNA processing is an underlying mechanism of amyotrophic lateral sclerosis (ALS). Missense mutations in a number of genes involved in RNA function and metabolisms are associated with ALS. Among these genes is angiogenin (ANG), the fifth member of the vertebrate-specific, secreted ribonuclease superfamily. ANG is an angiogenic ribonuclease, and both its angiogenic and ribonucleolytic activities are important for motor neuron health. Ribonuclease 4 (RNASE4), the fourth member of this superfamily, shares the same promoters with ANG and is co-expressed with ANG. However, the biological role of RNASE4 is unknown. To determine whether RNASE4 is involved in ALS pathogenesis, we sequenced the coding region of RNASE4 in ALS and control subjects and characterized the angiogenic, neurogenic, and neuroprotective activities of RNASE4 protein. We identified an allelic association of SNP rs3748338 with ALS and demonstrated that RNASE4 protein is able to induce angiogenesis in in vitro, ex vivo, and in vivo assays. RNASE4 also induces neural differentiation of P19 mouse embryonal carcinoma cells and mouse embryonic stem cells. Moreover, RNASE4 not only stimulates the formation of neurofilaments from mouse embryonic cortical neurons, but also protects hypothermia-induced degeneration. Importantly, systemic treatment with RNASE4 protein slowed weight loss and enhanced neuromuscular function of SOD1G93A mice.