Kazuto Nakae

A central role for Stat3 in IL-6-induced regulation of growth and differentiation in M1 leukemia cells.

Interleukin‐6 (IL‐6) induces either differentiation or growth of a variety of cells. Little is known about the molecular basis of this cellular decision. The family of signal transducer and activator of transcription (Stat) proteins are involved in signaling through a variety of cytokine and growth factor receptors, although their biological roles have not been established. To address whether Stat proteins play roles in IL‐6‐induced growth or differentiation, we introduced two types of mutant Stat3 acting in a dominant‐negative manner into M1 leukemic cells which respond to IL‐6 with growth arrest and terminal differentiation. We show that dominant‐negative forms of Stat3 inhibited both IL‐6‐induced growth arrest at G(0)/G1 and macrophage differentiation in the M1 transformants. Blocking of Stat activation resulted in inhibition of IL‐6‐induced repression of c‐myb and c‐myc. Furthermore, IL‐6 enhanced the growth of M1 cells primarily through shortening the length of the G1 period when Stat3 was suppressed. Thus IL‐6 generates both growth‐enhancing signals and growth arrest‐ and differentiation‐inducing signals at the same time. Stat3 may be a key molecule which determines the cellular decision from cell growth to differentiation in M1 cells.

Identification of a novel interleukin-6 response element containing an Ets-binding site and a CRE-like site in the junB promoter.

Interleukin-6 (IL-6) activation of the immediate-early gene junB has been shown to require both a tyrosine kinase and an unknown 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7)-sensitive pathway. Here we report the identification and characterization of an IL-6 immediate-early response element in the junB promoter (designated JRE-IL6) in HepG2 cells. The JRE-IL6 element, located at -149 to -124, contains two DNA motifs, an Ets-binding site (EBS) (CAGGAAGC) and a CRE-like site (TGACGCGA). Functional studies using variously mutated JRE-IL6 elements showed that both motifs were necessary and sufficient for IL-6 response of the promoter. The EBS of the JRE-IL6 element (JEBS) appears to bind a protein in the Ets family or a related protein which could also form a major complex with the EBSs of the murine sarcoma virus long terminal repeat or human T-cell leukemia virus type 1 long terminal repeat. The CRE-like site appears to weakly bind multiple CREB-ATF family proteins. Despite the similarity in the structure between the JRE-IL6 element and the polyomavirus enhancer PyPEA3, composed of an EBS and an AP1-binding site and known to be activated by a variety of oncogene signals, JRE-IL6 could not be activated by activated Ha-Ras, Raf-1, or 12-O-tetradecanoylphorbol-13-acetate. We show that IL-6 activates JRE-IL6 through an H7-sensitive pathway that does not involve protein kinase C, cyclic AMP-dependent kinase, Ca(2+)- or calmodulin-dependent kinases, Ras, Raf-1, or NF-IL6 (C/EBP beta). The combination of JEBS and the CRE-like site appears to form the basis for the selective and efficient response of JRE-IL6 to IL-6 signals, but not to signals generated by activated Ha-Ras, Raf-1, or protein kinase C.

ERM, a PEA3 subfamily of Ets transcription factors, can cooperate with c-Jun

A human cDNA clone for ERM, a member of the ets gene family, has been obtained by polymerase chain reaction with degenerate primers corresponding to highly conserved regions within an Ets DNA binding domain. ERM mRNA is expressed ubiquitously. The gene was mapped to chromosome 3q27. In in vivo transient-expression assays, ERM induced transcription more efficiently from a synthetic element containing both an ets-binding site (EBS) and a cyclic AMP response element (CRE) than from one containing an EBS alone. The activation of a synthetic EBS-CRE site by ERM was likely to involve a leucine zipper protein capable of dimerizing with CRE-BP1 leucine zipper. Indeed, ERM and c-Jun synergistically activated the EBS-CRE without making an apparent ternary complex. The synergy between c-Jun and ERM may be attributed to the enhancing effect of c-Jun on the transcription activity of ERM, because c-Jun increased ERM transcription activity by more than 20-fold in an assay system using a variety of fusion proteins between a Gal4 DNA-binding domain and a portion of ERM. This enhancing effect of c-Jun required the amino-terminal portion of ERM.

Cytokine effects on phagocytosis of rod outer segments by retinal pigment epithelial cells of normal and dystrophic rats

PURPOSE Phagocytosis of rod outer segments (ROS) is an important function of retinal pigment epithelial (RPE) cells. Since the details of the process are not fully known, we studied effects of cytokines produced by RPE and photoreceptor cells on phagocytosis of ROS by rat RPE cells. METHODS RPE cells were isolated and cultivated from two strains of rats: Sprague-Dawley (SD) rats with normal phagocytosis and Royal College of Surgeons (RCS) rats, which have genetic deficiencies in ROS phagocytosis. A double immunofluorescence staining technique was used to study the effects in vitro of several cytokines on phagocytosis of ROS. RESULTS We found that transforming growth factor beta-1 (TGF-beta 1) had dose-dependent effects on RPE cells of both strains of rat: at a concentration of 10 ng/ml, TGF-beta 1 significantly (p < 0.01) reduced total ROS (to 74% of control in SD rats and to 51% of control in RCS rats), reduced bound ROS (to 56% of control in SD rats and to 48% in RCS rats), and increased the ratio of ingested ROS to total ROS (to 140% in SD rats but not significantly in RCS rats). Treatment of medium with anti-TGF-beta 1 antibody before incubation of RPE cells of SD rats with TGF-beta 1 decreased the magnitude of these effects. The cytokine acidic fibroblast growth factor (aFGF, 10 ng/ml) affected RPE cells of SD rats only, decreasing ROS ingested to 56% of control and the ratio of ingested ROS to total ROS to 64% of control. We also examined effects of basic fibroblast growth factor and insulin-like growth factor. None of the cytokines tested increased ingestion of ROS by RPE cells of RCS rats. CONCLUSIONS Our results suggest that TGF-beta 1 and aFGF have roles in regulating ROS phagocytosis by normal and dystrophic RPE cells in the rat.

Complete epiretinal membrane separation in highly myopic eyes with retinal detachment resulting from a macular hole 1

PURPOSE Epiretinal membranes (ERMs) in highly myopic eyes may result in macular holes and subsequent retinal detachment. However, removing friable, thin ERMs from detached retinas is often difficult. We report the efficacy of a diamond-dusted silicone cannula in the removal of ERMs from detached retinas. METHODS Seventeen consecutive highly myopic eyes (16 patients) with retinal detachment underwent pars plana vitrectomy with gas tamponade. Peeling of the ERM adjacent to the macular hole was performed using either conventional tools (n = 11) or a diamond-dusted silicone cannula (n = 6). The rate of complete membrane peeling and the effect of membrane removal on the anatomic success rate were compared between groups. RESULTS Retinal reattachment occurred in 13 (92.9%) of the 14 eyes in which the ERM was removed completely; redetachment occurred in the other three eyes, with further surgical interventions in two eyes. The reattachment rate was significantly higher (P = .005) when ERM removal was complete than when there was residual ERM. In the initial surgery, the ERM was successfully removed in all 6 eyes (100%) in the diamond-dusted silicone cannula group and in 5 of 11 eyes (45.5%) in the conventional group (P = .041); the reattachment rate was 100% in the diamond-dusted silicone cannula group and 45.5% in the conventional group (P = .005). When a second surgery was performed, the use of the diamond-dusted silicone cannula was also better than conventional tools for removing the residual ERMs, resulting in retinal reattachment. CONCLUSION In highly myopic eyes with a macular hole and subsequent retinal detachment, complete ERM removal is closely related to successful retinal reattachment. The diamond-dusted silicone cannula appears to be more effective than conventional tools for removing ERM and may increase the anatomic success rate. Because of the limitations of a small series, a prospective, randomized trial is required to confirm the current beneficial results of using a diamond-dusted silicone cannula.

Complete epiretinal membrane separation in highly myopic eyes with retinal detachment resulting from a macular hole11A product based on the results of this study is commercially available as a diamond-dusted membrane scraper (DDMS, Synergetics, Inc, Chesterfield, Missouri, and the Inami Company, Tokyo, Japan). The authors have no financial or proprietary interest in any materials discussed in this report.

PURPOSE Epiretinal membranes (ERMs) in highly myopic eyes may result in macular holes and subsequent retinal detachment. However, removing friable, thin ERMs from detached retinas is often difficult. We report the efficacy of a diamond-dusted silicone cannula in the removal of ERMs from detached retinas. METHODS Seventeen consecutive highly myopic eyes (16 patients) with retinal detachment underwent pars plana vitrectomy with gas tamponade. Peeling of the ERM adjacent to the macular hole was performed using either conventional tools (n = 11) or a diamond-dusted silicone cannula (n = 6). The rate of complete membrane peeling and the effect of membrane removal on the anatomic success rate were compared between groups. RESULTS Retinal reattachment occurred in 13 (92.9%) of the 14 eyes in which the ERM was removed completely; redetachment occurred in the other three eyes, with further surgical interventions in two eyes. The reattachment rate was significantly higher (P = .005) when ERM removal was complete than when there was residual ERM. In the initial surgery, the ERM was successfully removed in all 6 eyes (100%) in the diamond-dusted silicone cannula group and in 5 of 11 eyes (45.5%) in the conventional group (P = .041); the reattachment rate was 100% in the diamond-dusted silicone cannula group and 45.5% in the conventional group (P = .005). When a second surgery was performed, the use of the diamond-dusted silicone cannula was also better than conventional tools for removing the residual ERMs, resulting in retinal reattachment. CONCLUSION In highly myopic eyes with a macular hole and subsequent retinal detachment, complete ERM removal is closely related to successful retinal reattachment. The diamond-dusted silicone cannula appears to be more effective than conventional tools for removing ERM and may increase the anatomic success rate. Because of the limitations of a small series, a prospective, randomized trial is required to confirm the current beneficial results of using a diamond-dusted silicone cannula.